title: Glycosylated SV2 and gangliosides as dual receptors for botulinum neurotoxin serotype F.
abstract: Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using alpha(1-3,4)-fucosidase, endo-beta-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain alpha2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a >> GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.
authors.affiliation: Department of Biochemistry, Medical Collegeof Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
authors.forename: Zhuji
authors.initials: Z
authors.lastname: Fu
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Barbieri
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
authors.forename: Michael R
authors.initials: MR
authors.lastname: Baldwin
mesh_terms: D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000378:metabolism; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000378:metabolism
chemical_list: D005732:Gangliosides; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C489618:Sv2a protein, rat; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096321:botulinum toxin type F