Abstract
Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.
Highlights
Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus Clostridium and are responsible for the deadly disease called botulism manifested by neuromuscular paralysis (Erbguth and Naumann, 1999; Schiavo et al, 2000; Montecucco and Molgó, 2005)
The neuronal specificity of the seven different BoNT serotypes is an active area of investigation, they all share a general mechanism of action, which has evolved to target the nervous system
One specific limitation is that the biochemical BINACLE assay takes into account only receptor binding and the vesicle-associated membrane proteins (VAMPs) cleavage steps of intoxication without probing the cellular translocation function and cannot fully replace the mouse bioassay in the quality control of pharmaceutical products
Summary
Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus Clostridium and are responsible for the deadly disease called botulism manifested by neuromuscular paralysis (Erbguth and Naumann, 1999; Schiavo et al, 2000; Montecucco and Molgó, 2005). A typical BoNT is expressed by bacteria as a single chain precursor protein that is processed into two polypeptide chains – a 100 kD heavy chain consisting of the receptor-binding domain and the translocation domain which is linked via a disulphide bond to 50 kD light chain, a SNARE protease (Lacy et al, 1998; Chaddock and Marks, 2006; Binz and Rummel, 2009). In order to reach their intraneuronal substrates, BoNTs first bind neuronal surface gangliosides and a synaptic vesicle protein (synaptotagmin or SV2) on the presynaptic membrane for subsequent internalization (Montecucco and Schiavo, 1994; Binz and Rummel, 2009). Once the internalized vesicle acidifies, the botulinum translocation domain changes conformation to form a putative protein transduction channel that enables translocation of the protease into the cytosol following reduction of the disulphide bond (Koriazova and Montal, 2003; Puhar et al, 2004; Pirazzini et al, 2013)
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