How botulinum and tetanus neurotoxins block neurotransmitter release

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.     

Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses

The clostridial neurotoxins responsible for tetanus and botulism are proteins consisting of three domains endowed with different functions: neurospecific binding, membrane translocation and proteolysis for specific components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular junction, is internalized and transported retroaxonally to the spinal cord. The spastic paralysis induced by the toxin is due to the blockade of neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven serotypes of botulinum neurotoxins (BoNTs) act at the periphery by inducing a flaccid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G cleave specifically at single but different peptide bonds, of the vesicle associated membrane protein (VAMP) synaptobrevin, a membrane protein of small synaptic vesicles (SSVs). BoNT types A, C and E cleave SNAP-25 at different sites located within the carboxyl-terminus, while BoNT type C additionally cleaves syntaxin. The remarkable specificity of BoNTs is exploited in the treatment of human diseases characterized by a hyperfunction of cholinergic terminals.     

Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons

The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.     

Tetanus and botulinum toxins are zinc metallopeptidases: molecular mechanisms and inhibition of their neurotoxicity

The very high toxicity of tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) are related to their nature of zinc metallopeptidases able to selectively cleave small proteins involved in neurotransmitters exocytosis. At this time, there is no efficient and selective therapy towards tetanos and tobulism as well as protection against a possible spreading of the toxins. We have therefore investigated the minimum sequences of TeNT and BoNT substrates allowing an efficient and simple fluorescent dosage of the enzymatic activity to be developed. Using synaptobrevin (93 amino acids) as substrate of TeNT and several fragments synthesised by solid phase method, we have shown that the clostridial neurotoxins behave as allosteric-type enzymes. This is the first example in zinc metallopeptidases. Based on these results a strategy, including the use of combinatorial chemistry, was carried out issuing in the design of the first potent inhibitors of TeNT and BoNT.     

Mechanism of action of tetanus and botulinum neurotoxins

The clostridial neurotoxins responsible for tetanus and botulism are metallo-proteases that enter nerve cells and block neurotransmitter release via zinc-dependent cleavage of protein components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular Junction and is internalized and transported retroaxonally to the spinal cord. Whilst TeNT causes spastic paralysis by acting on the spinal inhibitory interneurons, the seven serotypes of botullnum neurotoxins (BoNT) induce a flaccid paralysis because they intoxicate the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G specifically cleave VAMP/synaptobrevin, a membrane protein of small synaptic vesicles, at different single peptide bonds. Proteins of the presynaptic membrane are specifically attacked by the other BoNTs: serotypes A and E cleave SNAP-25 at two different sites located within the carboxyl terminus, whereas the specific target of serotype C is syntaxin.     

Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin

Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.     

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.     

Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism

The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.     

Comparative role of neurotoxin-associated proteins in the structural stability and endopeptidase activity of botulinum neurotoxin complex types A and E

Seven serotypes of botulinum neurotoxins, the most toxic substances known to mankind, are each produced by different strains of Clostridium botulinum along with a group of neurotoxin-associated proteins (NAPs). NAPs play a critical role in the toxicoinfection process of botulism in addition to their role in protecting the neurotoxin from proteolytic digestion in the GI tract as well as from adverse environmental conditions. In this study we have investigated the effect of temperature on the structural and functional stability of BoNT/A complex (BoNT/AC) and BoNT/E complex (BoNT/EC). Although the NAPs in the two complexes are quite different, both groups of NAPs activate the endopeptidase activities of their BoNTs without any need to reduce the disulfide bonds between light and heavy chains of respective BoNTs. BoNT/AC attains optimum enzyme activity at the physiological temperature of 37 degrees C whereas BoNT/EC is maximally active at 45 degrees C, and this is accompanied by conformational alterations in its polypeptide folding at this temperature, leading to favorable binding with its intracellular substrate, SNAP-25, and subsequent cleavage of the latter. BoNT/A in its complex form is found to be structurally more stable against temperature whereas BoNT/E in its complex form is functionally better protected against temperature. Based on the analysis of isolated NAPs we have observed that the structural stability of the BoNT/AC is contributed by the NAPs. In addition to the unique structural conditions in which the enzyme remains active, functional stability of botulinum neurotoxins against temperature plays a critical role in the survival of the agent in cooked food and in food-borne botulism.     

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.     

Membrane interactions of tetanus and botulinum neurotoxins: a photolabelling study with photoactivatable phospholipids

Tetanus and botulinum neurotoxins (TeNT and BoNT) bind strongly and specifically to the nervous tissue, as it can be inferred from their potency and from their effects restricted to the nervous system. The molecular basis of these properties are presently unknown. As a first approach, we have investigated the interaction of TeNT and BoNT with model membranes by photolabelling with phospholipid analogues carrying the photoreceptor group at different positions of the lipid molecule in order to probe different membrane regions. We found that at neutral pH TeNT and BoNTs (type A, B and E) adsorb onto the surface of negatively charged liposomes. Polysialogangliosides increase this interaction only slightly thus suggesting that they provide a minor contribution to toxin lipid binding. On this basis we propose that clostridial neurotoxins bind to lipids via both a predominant unspecific interaction with negatively charged lipids (including gangliosides) and a specific, but weaker, interaction with polysialogangliosides. At acidic pH values both chains of these neurotoxins are labelled strongly by photogroups located in the hydrophobic milieu of the membrane with a pH dependence that overlaps the range of pH values reached in the endosomal lumen. This result is consistent with their insertion into the lipid bilayer in agreement with the idea that clostridial neurotoxins may penetrate into cells via intracellular low pH compartments.     

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.     

Development of vaccines for prevention of botulism

Botulism is a potentially lethal disease caused by one of seven homologous neurotoxic proteins usually produced by the bacterium, Clostridium botulinum. This neuromuscular disorder occurs through an exquisite series of molecular events, ultimately ending with the arrest of acetylcholine release and hence, flaccid paralysis. The development of vaccines that protect against botulism dates back to the 1940s. Currently, a pentavalent vaccine that protects against BoNT serotypes A-E and a separate monovalent vaccine that protects against BoNT serotype F are available as Investigational New Drugs. However, due to the numerous shortcomings associated with the toxoid vaccines, several groups have efforts towards developing next-generation vaccines. Identifying a synthetic peptide that harbors a neutralizing epitope is one approach to a BoNT vaccine, while another employs the use of a Venezuelan equine encephalitis virus replicon vector to produce protective antigens in vivo against BoNT. The strategy used in our laboratory is to design synthetic genes encoding non-toxic, carboxy-terminal fragments of the C. botulinum neurotoxins (rBoNT(H(C))). The gene products are expressed in the yeast, Pichia pastoris, and purified to greater than 98% with yields typically ranging from 200-500 mg per kg of wet cells. Protective immunity to the purified products against high-level challenges of neurotoxin is elicited in mice and in non-human primates. A pre-Investigational New Drug meeting was held with the Food and Drug Administration, and the next milestone for the vaccine candidates will be clinical trials.     

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.     

Unique ganglioside recognition strategies for clostridial neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E … H … SXWY … G, with additional stabilizing interactions provided by two arginine residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.     

Insights into the different catalytic activities of Clostridium neurotoxins

The clostridial neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of botulinum neurotoxins (A-G) and tetanus toxin showed different toxicities and cleave their substrates with different efficiencies. However, the molecular basis of their different catalytic activities with respect to their substrates is not clear. BoNT/B light chain (LC/B) and TeNT light chain (LC/T) cleave vesicle-associated membrane protein 2 (VAMP2) at the same scissile bond but possess different catalytic activities and substrate requirements, which make them the best candidates for studying the mechanisms of their different catalytic activities. The recognition of five major P sites of VAMP2 (P7, P6, P1, P1', and P2') and fine alignment of sites P2 and P3 and sites P2 and P4 by LC/B and LC/T, respectively, contributed to their substrate recognition and catalysis. Significantly, we found that the S1 pocket mutation LC/T(K(168)E) increased the rate of native VAMP2 cleavage so that it approached the rate of LC/B, which explains the molecular basis for the lower k(cat) that LC/T possesses for VAMP2 cleavage relative to that of LC/B. This analysis explains the molecular basis underlying the VAMP2 recognition and cleavage by LC/B and LC/T and provides insight that may extend the pharmacologic utility of these neurological reagents.     

Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C–D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (∼two-third) and BoNT/D (∼one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.     

Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1

Botulinum neurotoxins (BoNTs), the causative agent of human botulism, are the most potent naturally occurring toxins known. BoNT/A1, the most studied BoNT, is also used as an important biopharmaceutical. In this study, the biological activity of BoNT/A1 is compared to that of BoNT/A2 using neuronal cell models. The data obtained indicate faster and increased intoxication of neuronal cells by BoNT/A2 than BoNT/A1, and that the mechanism underlying this increased toxicity is faster and more efficient cell entry that is independent of ganglioside binding. These results have important implications for the development of new BoNT based therapeutics and BoNT countermeasures.     

Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins

In botulism disease, neurotransmitter release is blocked by a group of structurally related neurotoxin proteins produced by Clostridium botulinum. Botulinum neurotoxins (BoNT, A-G) enter nerve terminals and irreversibly inhibit exocytosis via their endopeptidase activities against synaptic proteins SNAP-25, VAMP, and Syntaxin. Type A C. botulinum secretes the neurotoxin along with 5 other proteins called neurotoxin associated proteins (NAPs). Here, we report that hemagglutinin-33 (Hn-33), one of the NAP components, enhances the endopeptidase activity of not only BoNT/A but also that of BoNT/E, both under in vitro conditions and in rat synaptosomes. BoNT/A endopeptidase activity in vitro is about twice as high as that of BoNT/E under disulfide-reduced conditions. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E (which otherwise have only residual endopeptidase activity) enhanced their in vitro endopeptidase activity by 21- and 25-fold, respectively. Cleavage of rat-brain synaptosome SNAP-25 by BoNTs was used to assay endopeptidase activity under nerve-cell conditions. Reduced BoNT/A and BoNT/E cleaved synaptosomal SNAP-25 by 20% and 15%, respectively. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E enhanced their endopeptidase activities by 13-fold for the cleavage of SNAP-25 in synaptosomes, suggesting a possible functional role of Hn-33 in association with BoNTs. We believe that Hn-33 could be used as an activator in the formulation of the neurotoxin for therapeutic use.