Analysis of synaptic neurotransmitter release mechanisms using bacterial toxins

Several bacterial toxins are powerful and highly specific tools for studying basic mechanisms involved in cell biology. Whereas the clostridial neurotoxins are widely used by neurobiologists, many other toxins (i.e. toxins acting on small G-proteins or actin) are still overlooked. Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT), known under the generic term of clostridial neurotoxins, are characterized by their unique ability to selectively block neurotransmitter release. These proteins are formed of a light (Mr approximately 50) and a heavy (Mr approximately 100) chain which are disulfide linked. The cellular action of BoNT and TeNT involves several steps: heavy chain-mediated binding to the nerve ending membrane, endocytosis, and translocation of the light chain (their catalytic moiety) into the cytosol. The light chains each cleaves one of three, highly conserved, proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25 also termed SNAREs) implicated in fusion of synaptic vesicles with plasma membrane at the release site. Hence, when these neurotoxins are applied extracellularly, they can be used as specific tools to inhibit evoked and spontaneous transmitter release from certain neurones whereas, when the membrane limiting steps are bypassed by the mean of intracellular applications, BoNTs orTeNT can be used to affect regulated secretion in various cell types. Several members of the Rho GTPase family have been involved in intracellular trafficking of synaptic vesicles and secretory organelles. As they are natural targets for several bacterial exoenzymes or cytotoxins, their role in neurotransmitter release can be probed by examining the action of these toxins on neurotransmission. Such toxins include: i) the non permeant C3 exoenzymes from C. botulinum or C. limosum which ADP-ribosylate and thereby inactivate Rho, ii) exoenzyme S from Pseudomonas aeruginosa which ADP-ribosylates different members of the Ras, Rab, Ral and Rap families, iii) toxin B from C. difficile which glucosylates Rho, Rac and CDC42, iv) lethal toxin from C. sordellii which glucosylates Rac, Ras and to a lesser extent, Rap and Ral, but not on Rho or CDC42, and v) CNF deamidases secreted by pathogenic strains of E. coli which activate Rho and, to a lesser extent, CDC42. Since these toxins or exoenzymes have no or little ability to enter into the neurones, they must be applied intraneuronally to bypass the membrane limiting steps. Injection of several of these toxins into Aplysia neurones allowed us to reveal a new role for Rac in the control of exocytosis. ADP-ribosylating enzymes, which specifically act on monomeric actin (C2 binary toxin from C. botulinum and iota toxin from C. perfringens), are potential tools to probe the role of actin filaments during secretion.     

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.     

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 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.     

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.     

Internalization and Proteolytic Action of Botulinum Toxins in CNS Neurons and Astrocytes

Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins.     

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.     

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.     

Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis

Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.     

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.     

Retargeted clostridial neurotoxins as novel agents for treating chronic diseases

Botulinum neurotoxin (BoNT) A and B are used to treat neuropathic disorders; if retargeted, these agents could be used to treat medical conditions that involve secretion from nonneuronal cells. Here, we report novel strategies for successfully retargeting BoNTs, and also tetanus neurotoxin (TeNT), to primary human blood monocyte-derived macrophages where BoNT/B inhibited the release of tumor necrosis factor-α, a cytokine that plays a key role in inflammation. Furthermore, mice treated with retargeted BoNT/B exhibited a significant reduction in macrophage (MΦ) recruitment, indicating that these toxins can be used to treat chronic inflammation.     

Bafilomycin A1 Inhibits the Action of Tetanus Toxin in Spinal Cord Neurons in Cell Culture

Abstract: Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release. However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively. In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT. To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium-evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA). baf A1, which inhibits the vacuolar-type H⁺-ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose-dependent manner. TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 n M baf A1, respectively, compared with cultures treated with the inhibitor alone. The effects of baf A1 are fully reversible. In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action. These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1-sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.     

Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). To understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 A resolution. The structure of BoNT/G-LC reveals a C-terminal beta-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1' residue. In addition, structural and sequence analyses have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP.     

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.     

Multiple Domains of Tetanus Toxin Direct Entry into Primary Neurons

Tetanus toxin elicits spastic paralysis by cleaving VAMP‐2 to inhibit neurotransmitter release in inhibitory neurons of the central nervous system. As the retrograde transport of tetanus neurotoxin (TeNT) from endosomes has been described, the initial steps that define how TeNT initiates trafficking to the retrograde system are undefined. This study examines TeNT entry into primary cultured cortical neurons by total internal reflection fluorescence (TIRF) microscopy. The initial association of TeNT with the plasma membrane was dependent upon ganglioside binding, but segregated from synaptophysin1 (Syp1), a synaptic vesicle (SV) protein. TeNT entry was unaffected by membrane depolarization and independent of SV cycling, whereas entry of the receptor‐binding domain of TeNT (HCR/T) was stimulated by membrane depolarization and inhibited by blocking SV cycling. Measurement of the incidence of colocalization showed that TeNT segregated from Syp1, whereas HCR/T colocalized with Syp1. These studies show that while the HCR defines the initial association of TeNT with the cell membrane, regions outside the HCR define how TeNT enters neurons independent of SV cycling. This provides a basis for the unique entry of botulinum toxin and tetanus toxin into neurons.      

Traffic of botulinum toxins A and E in excitatory and inhibitory neurons

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma-aminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.     

Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (His(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. (His(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay.     

Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding-translocating heavy chain (HC; 100 kDa) linked through a disulfide bond. The complete sequence of BoNT/A consists of 1296 amino acid residues. The beta-trefoil domain for BoNT/A to which gangliosides bind starts at Ser 1092 and this fragment represents the C-half of the C-terminus of the heavy chain (C-quarter HC or HCQ). The recombinant HCQ DNA was successfully cloned into an expression vector (pET15b), which was used to transform Escherichia coli strain BL21-Star (DE3) for expression. Expression of HCQ was obtained by an extended post-induction time of 15 h at 30 degrees C. The recombinant histidine tagged HCQ protein was isolated and purified by nickel affinity gel column chromatography and its molecular weight was verified by gel electrophoresis. The HCQ was positively identified by antibodies raised against BoNT/A employing immunological dot-blot and Western blot assays. HCQ was shown to bind with synaptotagmin (a known BoNT/A receptor) and gangliosides, indicating that the expressed and purified HCQ protein retains a functionally active conformation.     

Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.     

A molecular basis underlying differences in the toxicity of botulinum serotypes A and E

Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K (SNAP-25), and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.