A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic.
ABSTRACT: Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT(A)) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT(A) was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC(A)) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT(A) activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC(E) to a BoNT(A) enzymically inactive mutant (BoTIM(A)) yielded a novel LC(E)-BoTIM(A) protein that targets neurons, and the BoTIM(A) moiety also delivers and stabilizes the inhibitory LC(E), giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT(E). LC(E-)BoTIM(A)(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT(E), reaffirming that these residues are critical for the persistent action of LC(E)-BoTIM(A) as well as BoNT(A). LC(E)-BoTIM(A) inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.
Project description:Botulinum neurotoxins (BoNTs), which are highly toxic proteins responsible for botulism, are produced by different strains of Clostridium botulinum. These various strains of bacteria produce seven distinct serotypes, labeled A-G. Once inside cells, the zinc-dependent proteolytic light chain (LC) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis, specifically synaptosomal-associated protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis, detection limits, and cost. SNAPtide, a 13-amino acid fluorescence resonance energy transfer (FRET) peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage. Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity, reducing the apparent signal. This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased. In this study, fluorescence internal quenching (FIQ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures. The BoNT/A LC endopeptidase kinetics at the optimally active temperature (37 °C) using SNAPtide were studied and used to demonstrate the FIQ correction factors in this study. The FIQ correction factors developed provide a convenient method to allow for improved accuracy in determining and comparing BoNT/A LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures.
Project description:Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C S51T/R52N/N53P (BoNT/C ?-51) and BoNT/C L200W/M221W/I226W (BoNT/C ?-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C in vivo. Although BoNT/C ?-51 and BoNT/C ?-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C ?-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C ?-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.
Project description:Botulinum neurotoxin A (BoNT/A) is one of the most deadly toxins and is the etiological agent of the potentially fatal condition, botulism. Herein, we investigated 8-hydroxyquinoline (quinolin-8-ol) as a potential inhibitor scaffold for preventing the deadly neurochemical effects of the toxin. Quinolinols are known chelators that can disrupt the BoNT/A metalloprotease zinc-containing active site, thus impeding its proteolysis of the endogenous protein substrate, synaptosomal-associated protein 25 (SNAP-25). By use of this information, the structure-activity relationship (SAR) of the quinolinol-5-sulfonamide scaffold was explored through preparation of a crude sulfonamide library and evaluation of the library in a BoNT/A LC enzymatic assay. Potency optimization of the sulfonamide hit compounds was undertaken as informed by docking studies, granting a lead compound with a submicromolar Ki. These quinolinol analogues demonstrated inhibitory activity in a cell-based model for SNAP-25 cleavage and an ex vivo assay for BoNT/A-mediated muscle paralysis.
Project description:Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960's, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a K(i) of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.
Project description:Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an ?-exosite binding probe. Experiments with this unique probe demonstrated that ?-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why ?-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.
Project description:Botulinum neurotoxins (BoNTs) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well-understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs) are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1(+) pre-synaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT.
Project description:Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle(202)]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH(2) acetylated at positions 156 to 203 [Nop(197), Pya(200), Nle(202)]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 x 10(6) M(-1) s(-1) and 8.85 x 10(6) M(-1) s(-1), respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.
Project description:Botulinum neurotoxins (BoNT/A-G) act by blocking synaptic vesicle exocytosis. Whether BoNTs disrupt additional neuronal functions has not been addressed. Here we report that cleavage of syntaxin 1 by BoNT/C, and cleavage of SNAP-25 by BoNT/E both induce degeneration of neurons. Furthermore, although SNAP-25 cleaved by BoNT/A still supports neuron survival, it has reduced capacity to tolerate additional mutations. We demonstrate that syntaxin 1 and SNAP-25 cooperate as SNARE proteins to support neuron survival. Exogenous expression of other homologous SNARE proteins, syntaxin 2/3/4 and SNAP-23, which are resistant to BoNT/C and E in neurons, can substitute syntaxin 1/SNAP-25 and prevent toxin-induced neuron death. Finally, we find that neuronal death is due to blockage of plasma membrane recycling processes that utilize syntaxin 1/SNAP-25, independent of synaptic vesicle exocytosis. These findings establish neuronal cytotoxicity for BoNTs and reveal syntaxin 1/SNAP-25 as the ubiquitous and essential SNARE proteins mediating multiple fusion events on neuronal plasma membranes.
Project description:Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.
Project description:Botulinum neurotoxins (BoNTs) are highly potent toxins that cleave neuronal SNARE proteins required for neurotransmission, causing flaccid paralysis and death by asphyxiation. Currently, there are no clinical treatments to delay or reverse BoNT-induced blockade of neuromuscular transmission. While aminopyridines have demonstrated varying efficacy in transiently reducing paralysis following BoNT poisoning, the precise mechanisms by which aminopyridines symptomatically treat botulism are not understood. Here we found that activity-dependent potentiation of presynaptic voltage-gated calcium channels (VGCCs) underlies 3,4-diaminopyridine (3,4-DAP)-mediated rescue of neurotransmission in central nervous system synapses and mouse diaphragm neuromuscular junctions fully intoxicated by BoNT serotype A. Combinatorial treatments with 3,4-DAP and VGCC agonists proved synergistic in restoring suprathreshold endplate potentials in mouse diaphragms fully intoxicated by BoNT/A. In contrast, synapses fully intoxicated by BoNT serotypes D or E were refractory to synaptic rescue by any treatment. We interpret these data to propose that increasing the duration or extent of VGCC activation prolongs the opportunity for low-efficiency fusion by fusogenic complexes incorporating BoNT/A-cleaved SNAP-25. The identification of VGCC agonists that rescue neurotransmission in BoNT/A-intoxicated synapses provides compelling evidence for potential therapeutic utility in some cases of human botulism.