Substrate residues N‐terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity

Wictome, M; Rossetto, Ornella; Montecucco, Cesare; Shone, Clifford C.

doi:10.1016/0014-5793(96)00431-0

Cited by 36 publications

(34 citation statements)

References 18 publications

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“…Recognition within the cleavage region of SNAP-25 involves four pockets within LC/A: the S5, S3, S1Ј, and S4Ј pockets (21), whereas recognition of the cleavage region of SNAP-25 involves three pockets within LC/E: the S3, S2, and S1Ј pockets (22). Thus, although sharing a common mechanism, substrate recognition by LC/A and LC/E follow unique steps to enhance cleavage efficiency.BoNT serotypes and TeNT that cleave VAMP-2 also recognize extended regions of the substrate for efficient cleavage (16,18,(23)(24)(25)(26)). In the current study, BoNT/B and TeNT, which share ϳ51% primary amino acid identity and ϳ68% similarity and cleave VAMP-2 at the same site, were used to study VAMP-2 recognition and cleavage.…”

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Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

Chen

Hall

Barbieri

2008

Journal of Biological Chemistry

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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 (>32-fold), whereas mutations at P7, P4, P1, and P2 residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (>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.The botulinum neurotoxins (BoNTs) 2 are the most potent protein toxins for humans (1). BoNTs have been used for malicious purposes (2) but also relieve numerous human neurological afflictions, such as blepharospasm, in addition to being used for cosmetic enhancement (3-5). Recent studies provide a more detailed understanding of BoNT action, which should provide insight for developing novel products and therapies to treat human suffering.BoNTs are zinc proteases that cleave SNARE proteins to block synaptic vesicle fusion and neurotransmitter release to elicit flaccid paralysis. BoNTs are 150-kDa dichain proteins with AB structure-function properties; the N terminus encodes the zinc protease domain (light chain (LC)), and the C terminus encodes a translocation domain and a receptor binding domain (6, 7). The seven serotypes of BoNTs, termed A-G, are based upon antisera neutralization properties (8). Each BoNT serotype cleaves one of three neuronal SNARE proteins: SNAP-25, VAMP-2, or syntaxin 1a, except for BoNT/C, which cleaves both SNAP-25 and syntaxin 1a. BoNT/A and BoNT/E cleave SNAP-25 at different sites, whereas BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave VAMP-2 at distinct sites (9 -12). In addition, tetanus neurotoxin (TeNT) shares structural homology with the BoNTs and cleaves VAMP-2 at the same site as BoNT/B. Despite this similarity, TeNT elicits spastic paralysis due to the ability to retrograde traffic within the peripheral neurons to target interneurons (6, 13-15).Unlike other zinc proteases, BoNTs and TeNT rec...

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“…BoNT serotypes and TeNT that cleave VAMP-2 also recognize extended regions of the substrate for efficient cleavage (16,18,(23)(24)(25)(26). In the current study, BoNT/B and TeNT, which share ϳ51% primary amino acid identity and ϳ68% similarity and cleave VAMP-2 at the same site, were used to study VAMP-2 recognition and cleavage.…”

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confidence: 99%

Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

Chen

Hall

Barbieri

2008

Journal of Biological Chemistry

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“…Our first goal was to construct a yeast strain that could be killed by a BoNT͞LC endoprotease through cleavage of intracellular SNARE protein. For our initial analysis, B-LC was examined, because its substrate specificity has been extensively studied by reverse genetic approaches combined with cell-free protease assays (3,8,(15)(16)(17). B-LC recognizes Sb in human motor neurons and cleaves this SNARE protein at a specific QF bond, where Q represents the P1 residue at the N-terminal side of the cleavage site and F represents the PЈ1 residue at the C-terminal side of cleavage site (see Fig.…”

Section: Resultsmentioning

confidence: 99%

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

Fang

Luo

Henkel

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The seven functionally distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are dichains consisting of light chain (LC) with zinc-dependent endoprotease activity connected by one disulfide bond to heavy chain with neuronal-cell translocation and receptorbinding domains. LC-mediated proteolysis of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and consequent inhibition of synaptic vesicle fusion to the presynaptic membrane of human motor neurons are responsible for flaccid paralysis associated with botulism. LC endoproteolysis is complex, requiring highly extended SNARE sequences at the surface of intracellular membranes and prompting our development of a genetically amenable assay to monitor the interaction between BoNT͞LC and its SNARE substrate. Using BoNT serotype B as a model, the assay employs a chimeric SNARE protein where a portion of neuronal synaptobrevin (Sb) is fused to Snc2p, a Sb ortholog required for protein secretion from yeast cells. Regulated expression of serotype B-LC in yeast leads to cleavage of the chimera and a conditional growth defect. To assess utility of this assay for monitoring SNARE protein cleavage, we growth-selected chimeric SNARE mutations that inhibited proteolysis. When these mutations were introduced into Sb and examined for cleavage, substrate residues located near and distal to the cleavage site were important, including residues positioned near the Sb transmembrane domain, an unexplored aspect of BoNT cell intoxication. Additional mutations were positioned in a nine-residue SNARE motif, supporting a previously assigned role for this motif in LC recognition and providing proof of principle for the application of yeast-based technology to study intracellular BoNT͞LC endoproteases.substrate specificity ͉ endoprotease ͉ endopeptidase ͉ synaptic vesicle B otulinum neurotoxin (BoNT) intoxicates motor neurons at neuromuscular junctions to produce a generalized flaccid paralysis that is often fatal at extremely low doses of the toxin: LD 50 values of 1-5 ng͞kg in mice (1). The potency of BoNT has caused its listing as a class A select toxin by the Centers for Disease Control and Prevention, the National Institute of Allergy and Infectious Disease, and the U.S. Department of Agriculture. There are seven structurally related BoNT serotypes (A-G), each possessing a heavy chain capable of transporting its light chain (LC) partner into human neuronal cells. Upon entering the cytoplasmic compartment, LC endoprotease cleaves and inactivates one of three membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including one vesicle SNARE, synaptobrevin (Sb), and two target membrane SNAREs, SNAP-25 and syntaxin. These SNARE proteins physically interact in a calcium-dependent manner, leading to fusion of synaptic vesicles to the presynaptic membrane of motor neurons (Fig. 1a). BoNT͞LC disables the physical interaction between SNARE proteins, inhibiting vesicle fusion and acetylcholine release into the n...

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“…Likewise, the action of BoNT B does not require Ca2~in the incubation buffer (Ashton and Dolly, 1991), whereas the efficacy of TeTX action is attenuated under such conditions (Ashton et a!., 1995). Clearly, the detailed actions of BoNT B and TeTX differ, as does the size of Sbr peptide required to allow proteolytic cleavage by these two toxins , a result that may be due to the toxins interacting with different motifs on this substrate (Wictome et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Microtubules and Microfilaments Participate in the Inhibition of Synaptosomal Noradrenaline Release by Tetanus Toxin

Ashton

Dolly

1997

Journal of Neurochemistry

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Tetanus toxin (TeTX) has been demonstrated to inhibit transmitter release by two mechanisms: Zn 2~-dependent proteolytic cleavage of synaptobrevin and activation of a neuronal transglutaminase. Herein, attenuation of TeTX-induced blockade of noradrenaline release from synaptosomes was achieved by prior disassembly of microfilaments with cytochalasin D or breakdown of microtubules by colchicine or nocodazole. These drugs and monodansylcadaverine, a transglutaminase inhibitor, displayed some additivity in antagonizing the inhibitory effect of the toxin on synaptosomal transmitter release; as none of them reduced synaptobrevin cleavage, all appear to work independently of the toxin's proteolytic action. Prior stabilization of microtubules with taxol prevented the antagonism seen with coichicine, highlighting that this cytoskeletal component is the locus of the effect of coichicine. Replacement of Ca2~with Ba2~caused disappearance of the fraction of evoked secretion whose inhibition by TeTX is reliant on polymerized actin but did not alter the blockade by toxin that is dependent on microtubules. Two temporally distinguished phases of release were reduced by TeTX, and colchicine lessened its effects on both. Blockade of the fast phase ( 10 s) of secretion by TeTX was unaffected by cytochalasin D, but it clearly antagonized the toxin-induced inhibition of the slow (10-s to 5-min) component; it is notable that such antagonism was accentuated during a second bout of evoked release. These findings are consistent with sustained release requiring dissociation of synaptic vesicles from the microfilaments, a step that seems to be perturbed by TeTX.

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Substrate residues N‐terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity

Cited by 36 publications

References 18 publications

Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

Microtubules and Microfilaments Participate in the Inhibition of Synaptosomal Noradrenaline Release by Tetanus Toxin

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