Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F

Abstract

Botulinum neurotoxin causes rapid flaccid paralysis through 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 α(1-3,4)- fucosidase, endo-β-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 α2, 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 recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.

Dynamin Inhibition Blocks Botulinum Neurotoxin Type A Endocytosis in Neurons and Delays Botulism

“…Five-minute depolarization led to 59% of Alexa Fluor 488-BoNT/A-Hc positive vesicles co-localizing with VAMP2-positive puncta (Fig. 2), in agreement with the results of previous studies (17,18). Interestingly, 34% of Alexa Fluor 488-BoNT/A-Hc/VAMP2 dual-positive compartments also partially or fully co-localized with Rab5-positive compartments, suggesting that Alexa Fluor 488-BoNT/A-Hc can at least partially traffic through synaptic vesicles and early endosomes in hippocampal neurons (Fig.…”

“…The neurons were allowed to mature for at least 14 days in vitro before use. Neurons were removed from the co-culture and incubated for 5 min at 37°C with 100 nM Alexa Fluor 488-BoNT/A-Hc in a low K ϩ buffer (15 mM HEPES, 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl 2 , 0.5 mM MgCl 2 , 5.6 mM D-glucose, 0.5 mM ascorbic acid, 0.1% bovine serum albumin (BSA), pH 7.4) or high K ϩ buffer (modified to contain 95 mM NaCl and 56 mM KCl) (18), with or without Dyngo-4a or Dynasore as indicated. The cells were fixed with 4% paraformaldehyde, processed for immunocytochemistry (33), imaged (LSM510 confocal microscope; Zeiss), and analyzed using Zen software (Zeiss) or LaserPix (Bio-Rad).…”

“…BoNTs have previously been reported to enter hippocampal neurons in an activity-dependent manner (17,18). To validate our probe, hippocampal neurons were incubated with Alexa Fluor 488-BoNT/A-Hc in low or depolarizing high K ϩ buffer, then fixed and processed for immunocytochemistry.…”

“…The heavy chain is divided into two functionally distinct regions: a binding (Hc) and a translocation (H N ) domain (11). The binding domain initially interacts with low affinity to a group of gangliosides on the presynaptic plasma membrane (12), after which it binds to a protein acceptor (13)(14)(15)(16)(17)(18). The neurotoxin is subsequently internalized into an acidic compartment, prompting the translocation domain to create a pore and promoting the release of the Lc into the cytosol following hydrolysis of the disulfide bond (19).…”

Enhancing the Protective Immune Response against Botulism

“…Data collection and processing statistics are summarized in Table 1. The structure of HCR/A(W1266A) was solved by the molecular replacement method using the PHASER (39) program and using the structure of HCR/A (residues 875 to 1,295; protein data bank [PDB] code 3FUO) (40) as the search model. The initial structure obtained from molecular replacement trials was refined using the program CNS (41).…”