Abstract

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Highlights

  • Botulinum neurotoxins (BoNTs), which are produced by the bacteria Clostridium botulinum, are some of the most potent natural toxins

  • We found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C

  • The binding affinities of BoNT type C (BoNT/C) for gangliosides are first reported in the present study, its binding specificities had been analyzed previously using a thin layer chromatography overlay assay [12,24]

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Summary

Introduction

Botulinum neurotoxins (BoNTs), which are produced by the bacteria Clostridium botulinum, are some of the most potent natural toxins. All BoNTs cause flaccid paralysis by inhibiting the release of acetylcholine from peripheral neuromuscular junctions [2]. BoNT type C (BoNT/C) is synthesized as a single-chain polypeptide (approximately 150 kDa) that is proteolytically activated into a light chain (50 kDa) and a heavy chain (100 kDa). Cleavage of SNARE proteins, regardless of type, inhibits neurotransmitter release, because their assembly is required for neuroexocytosis. All serotypes of BoNT, except for BoNT/C, recognize both gangliosides and synaptic vesicle proteins as receptors for entering neuronal cells [6,7,8,9,10,11]. Gangliosides, but not synaptic vesicle proteins, are predicted to play a significant role in the recognition of neuronal cells by BoNT/C, because BoNT/C toxicity is decreased considerably in ganglioside-deficient mice [12]. Recent studies revealed that BoNT/C interacts with gangliosides at two binding sites, termed the ganglioside-binding pocket 2 (GBP-2) and sialic acid binding site 1 (Sia-1), and their preferences for gangliosides are different [13,14]

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