Botulinum Neurotoxin D Uses Synaptic Vesicle Protein SV2 and Gangliosides as Receptors

Abstract

Botulinum neurotoxins (BoNTs) include seven bacterial toxins (BoNT/A-G) that target presynaptic terminals and act as proteases cleaving proteins required for synaptic vesicle exocytosis. Here we identified synaptic vesicle protein SV2 as the protein receptor for BoNT/D. BoNT/D enters cultured hippocampal neurons via synaptic vesicle recycling and can bind SV2 in brain detergent extracts. BoNT/D failed to bind and enter neurons lacking SV2, which can be rescued by expressing one of the three SV2 isoforms (SV2A/B/C). Localization of SV2 on plasma membranes mediated BoNT/D binding in both neurons and HEK293 cells. Furthermore, chimeric receptors containing the binding sites for BoNT/A and E, two other BoNTs that use SV2 as receptors, failed to mediate the entry of BoNT/D suggesting that BoNT/D binds SV2 via a mechanism distinct from BoNT/A and E. Finally, we demonstrated that gangliosides are essential for the binding and entry of BoNT/D into neurons and for its toxicity in vivo, supporting a double-receptor model for this toxin.

Author Summary

BoNTs are a family of seven bacterial toxins (BoNT/A-G). Among the seven BoNTs, whether BoNT/D uses the same entry pathways and similar receptor-binding strategies as other BoNTs is not known. Previous studies have suggested that BoNT/D does not need a protein receptor nor ganglioside co-receptor, in contrast to all other BoNTs. Here we demonstrate that BoNT/D uses synaptic vesicle protein SV2 as its protein receptor and gangliosides as co-receptor, thus supporting the “double-receptor” model as a central theme for this class of toxins. Furthermore, we found that BoNT/D utilizes a SV2 binding mechanism distinct from BoNT/A and BoNT/E, two other BoNTs that use SV2 as receptors. This indicates that different BoNTs can develop their distinct mechanisms to target a common receptor protein.