Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A.

Kurazono, Hisao; Mochida, Sumiko; Binz, Thomas; Eisel, Ulrich; Quanz, Martin; Grebenstein, Oliver; Wernars, K.; Poulain, Bernard; Tauc, L; Niemann, Heiner

doi:10.1016/s0021-9258(18)42100-x

Cited by 85 publications

(6 citation statements)

References 51 publications

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“…If a destabilizing amino acid at the N‐terminus of a toxin were sufficient to make the protein unstable in the cytosol, one would expect that throughout evolution there would have been a selection against destabilizing amino acids at the N‐termini of toxic proteins that reach the cytosol. Therefore, we investigated the published N‐termini of some of the toxin domains that are believed to enter the cytosol (DeLange et al ., 1979; Lamb et al ., 1985; Nicosia et al ., 1986; Calderwood et al ., 1987; Yamamoto et al ., 1987; Escuyer et al ., 1988; Strockbine et al ., 1988; Bragg and Robertson, 1989; Ogata et al ., 1990; Krieglstein et al ., 1991; Wood et al ., 1991; Kurazono et al ., 1992; Huguet Soler et al ., 1996), but we could not find any systematic exclusion of destabilizing residues at the N‐termini of the toxin domains. Although it cannot be formally excluded that minor modifications of the N‐termini of these proteins occur upon translocation, the data suggest that placing a destabilizing residue at the N‐terminus may often not be sufficient to make a protein unstable.…”

Section: Discussionmentioning

confidence: 99%

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

Falnes

Olsnes

1998

The EMBO Journal

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The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the Nend rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for Nend rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed β-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process.

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Section: Discussionmentioning

confidence: 99%

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

Falnes

Olsnes

1998

The EMBO Journal

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“…Technical difficulties hitherto in expressing the L chain of BoNT/A in a heterologous host and subsequent isolation of the viable protein have limited molecular studies to those in the sea hare, Aplysia californica, where microinjection of L chain mRNA into the buccal ganglionic neurons caused a blockade of acetylcholine release (Kurazono et al, 1992). This technique allowed the importance of certain Nand C-terminal residues to be established from the observed loss of the neuroparalytic activity resulting from injection of mRNA encoding L chain deletion mutants.…”

Section: Discussionmentioning

confidence: 99%

Expression and Purification of the Light Chain of Botulinum Neurotoxin A: A Single Mutation Abolishes Its Cleavage of SNAP-25 and Neurotoxicity after Reconstitution with the Heavy Chain

Zhou

Paiva²,

Liu³

et al. 1995

Biochemistry

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Botulinum neurotoxin type A (BoNT/A) selectively and irreversibly inhibits acetylcholine release from peripheral nerve endings. While the toxin's heavy (H) chain contributes to neuronal binding and internalization, its light (L) chain is a Zn(2+)-dependent endoprotease that intracellularly cleaves synaptosomal-associated protein of M(r) = 25 kDa (SNAP-25). For research and clinical exploitation of this uniquely-acting neurotoxin, recombinant wild-type L chain was produced together with a mutant in which His227 in the Zn(2+)-binding motif was substituted by Tyr. The PCR-amplified wild-type and mutant L chain genes were cloned, fused to the gene for maltose-binding protein, and expressed at high levels in Escherichia coli. The soluble fusion proteins were purified using amylose affinity chromatography, and, after factor Xa cleavage, the free L chains were isolated. The wild-type was shown to proteolyze SNAP-25 at a rate approaching that of the native chain while the mutant was inactive. Reconstitution of the pure wild-type L chain with native homogeneous H chain yielded a disulfide-linked dichain form that inhibited neuromuscular transmission in vitro and produced the symptoms of botulism in vivo. After reconstitution with the H chain, the Tyr227 mutant L chain failed to show any neuroparalytic activity in either of these assays. This methodology allows, for the first time, routine preparation of recombinant forms of the L chain that are needed to decipher the molecular details of its interaction with substrate and, thereby, assist the design of effective inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The catalytic activity of CNTs was discovered when the determination of their primary structure (Minton 1995) revealed the presence of the His-Glu-Xaa-Xaa-His zinc-binding zinc-endopeptidases motif (Kurazono et al 1992;Schiavo et al 1992b,c;Wright et al 1992). Building upon this observation, it was soon demonstrated that TeNT was blocking ACh release at synapses of the buccal ganglion of Aplysia californica via a zinc-dependent protease activity (Schiavo et al 1992a,b).…”

Section: Zinc-endopeptidase Activitymentioning

confidence: 99%

Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses

Pellizzari

Rossetto

Schiavo

et al. 1999

Phil. Trans. R. Soc. Lond. B

261

197

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The clostridial neurotoxins responsible for tetanus and botulism are proteins consisting of three domains endowed with di¡erent functions: neurospeci¢c binding, membrane translocation and proteolysis for speci¢c 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 £accid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G cleave speci¢cally at single but di¡erent 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 di¡erent sites located within the carboxyl-terminus, while BoNT type C additionally cleaves syntaxin. The remarkable speci¢city of BoNTs is exploited in the treatment of human diseases characterized by a hyperfunction of cholinergic terminals.

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Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A.

Cited by 85 publications

References 51 publications

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

Expression and Purification of the Light Chain of Botulinum Neurotoxin A: A Single Mutation Abolishes Its Cleavage of SNAP-25 and Neurotoxicity after Reconstitution with the Heavy Chain

Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses

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