Abstract
Botulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells.
Highlights
The LC acts as a protease in neurons to cleave a set of proteins: BoNT/A, C and E cleave at distinct sites on a peripheral membrane protein known as SNAP-25; BoNT/B, D, F and G cleave at different sites on homologous vesicle proteins VAMP1, 2 and 3; and BoNT/C cleaves the plasma membrane protein syntaxin 1
Incubation with X-LC generated a single dominant peptide peak with a molecular weight of 2,063.1, which fits only A67-R86 of VAMP2 (Supplementary Fig. 3). These results demonstrate that X-LC has a single cleavage site on VAMP2 between R66 and A67
We found that X-LC cleaved both purified glutathione S-transferase (GST)-tagged cytoplasmic domain of VAMP4 (Fig. 2i), as well as native VAMP4 in brain detergent extracts (BDE) (Fig. 2j)
Summary
The LC acts as a protease in neurons to cleave a set of proteins: BoNT/A, C and E cleave at distinct sites on a peripheral membrane protein known as SNAP-25; BoNT/B, D, F and G cleave at different sites on homologous vesicle proteins VAMP1, 2 and 3 (vesicle-associated membrane proteins); and BoNT/C cleaves the plasma membrane protein syntaxin 1. These proteins are prototypes of the SNARE (soluble NSF attachment protein receptor) protein family, whose members mediate various membrane fusion events in eukaryotic cells[7,8]. A recent survey of infant botulism cases reported that B8% isolates are BoNT/A(B) strains[29]
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