Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner.
The extraordinarily high toxicity of botulinum neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum neurotoxin) serotypes A-G inhibit acetylcholine release leading to flaccid paralysis. Uptake of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is known about the cell entry mechanisms of the serotypes C and D, which display the lowest amino acid sequence identity compared with the other five serotypes. In the present study we demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by employing phrenic nerve hemidiaphragm preparations derived from mice expressing the gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI-XIV]. High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants identified two carbohydrate-binding sites. One site is located at a position previously identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).
Strotmeier
Jasmin
J
Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover, Germany.
Lee
Kwangkook
K
Völker
Anne K
AK
Mahrhold
Stefan
S
Zong
Yinong
Y
Zeiser
Johannes
J
Zhou
Jie
J
Pich
Andreas
A
Bigalke
Hans
H
Binz
Thomas
T
Rummel
Andreas
A
Jin
Rongsheng
R
D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018360:Crystallography, X-Ray; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009474:Neurons / Q000187:drug effects* / Q000473:pathology; D010446:Peptide Fragments / Q000737:chemistry; D010791:Phrenic Nerve / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D002241:Carbohydrates; D005732:Gangliosides; D050505:Mutant Proteins; D010446:Peptide Fragments; C096325:botulinum toxin type D; D001905:Botulinum Toxins; D019158:N-Acetylneuraminic Acid