Recombinant and Truncated Tetanus Neurotoxin Light Chain: Cloning, Expression, Purification, and Proteolytic Activity

Tonello, Fiorella; Pellizzari, Rossella; Pasqualato, Sebastiano; Grandi, Guido; Peggion, Evaristo; Montecucco, Cesare

doi:10.1006/prep.1998.1007

Cited by 15 publications

(7 citation statements)

References 16 publications

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“…The digestion was carried out in a buffer system (see Materials and methods), which has been previously shown to support the enzymatic activity of LC. 17,21 The density of the 18 kDa mouse brain VAMP-1 band, detected on anti-VAMP-1 Western blots, deteriorates as a function of the duration of incubation with AdLC PC12 lysates, but not AdLacZ PC12 lysates ( Figure 2a). Reprobing the same blot with an anti-LC antibody revealed a consistent level of LC in AdLC lysates.…”

Section: Lc Expression Induces Time-and Concentrationdependent Vamp-1mentioning

confidence: 99%

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

et al. 2004

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Clostridial neurotoxins have assumed increasing importance in clinical application. The toxin's light chain component (LC) inhibits synaptic transmission by digesting vesicle-docking proteins without directly altering neuronal health. To study the properties of LC gene expression in the nervous system, an adenoviral vector containing the LC of tetanus toxin (AdLC) was constructed. LC expressed in differentiated neuronal PC12 cells was shown to induce time-and concentration-dependent digestion of mouse brain synaptobrevin in vitro as compared to control transgene products. LC gene expression in the rat lumbar spinal cord disrupted hindlimb sensorimotor function in comparison to control vectors as measured by the Basso-Beattie-Bresnahan (BBB) scale (Po0.001) and rotarod assay (Po0.003).Evoked electromyography (EMG) showed increased stimulus threshold and decreased response current amplitude in LC gene-transferred rats. At the peak of functional impairment, neither neuronal TUNEL staining nor reduced motor neuron density could be detected. Spontaneous functional recovery was observed to parallel the cessation of LC gene expression. These results suggest that light chain gene delivery within the nervous system may provide a nondestructive means for focused neural inhibition to treat a variety of disorders related to excessive synaptic activity, and prove useful for the study of neural circuitry.

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Section: Lc Expression Induces Time-and Concentrationdependent Vamp-1mentioning

confidence: 99%

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

et al. 2004

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“…Nor do the present data formally prove the existence of a radial assembly of SNARE complexes only, as other proteins could be implicated in the interaction with R206 and D253 within the NMJ. However, the present data are strictly pertinent to the studies using cell-free membrane fusion model systems (Ellena et al, 2009;Gao et al, 2012;Hua and Scheller, 2001;Shi et al, 2012;Pobbati et al, 2006;Lu et al, 2008;Domanska et al, 2009;Giraudo et al, 2009;Karatekin et al, 2010;van den Bogaart et al, 2011;Diao et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…The presence of the mutations was confirmed by DNA sequencing. The mutated constructs were transformed into E. coli BL21 DE3 and the recombinant proteins expressed as GST fusion proteins and isolated as previously described (Tonello et al, 1999). Briefly, expression of the GST fusion proteins was induced for 3 hours at 30˚C with 1 mM IPTG (isopropyl-b-Dthiogalactoside) and proteins were isolated on a glutathione-Sepharose 4B affinity column (Amersham Biosciences) according to the manufacturer's instructions.…”

Section: Construction Of the Super Snare Complex Radial Modelmentioning

confidence: 99%

“…Recombinant syntaxin 1A cytoplasmic domain (1-261) and VAMP2 cytoplasmic domain (1-96), a kind gift from B. Davletov (LMB, Cambridge, UK), were expressed and purified as described previously (Hu et al, 2002). WILDTYPE was expressed and purified as detailed before (Tonello et al, 1999). The size of the PCR products were 119 bp in the case of SNAP-25 isoforms, 109 bp for syntaxin isoforms and 164 bp for rp49 (housekeeping gene).…”

Section: Construction Of the Super Snare Complex Radial Modelmentioning

confidence: 99%

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Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction

Megighian

Zordan

Pantano

et al. 2013

Journal of Cell Science

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SummaryThe SNARE proteins VAMP/synaptobrevin, SNAP-25 and syntaxin are core components of the apparatus that mediates neurotransmitter release. They form a heterotrimeric complex, and an undetermined number of SNARE complexes assemble to form a super-complex. Here, we present a radial model of this nanomachine. Experiments performed with botulinum neurotoxins led to the identification of one arginine residue in SNAP-25 and one aspartate residue in syntaxin (R206 and D253 in Drosophila melanogaster). These residues are highly conserved and predicted to play a major role in the protein-protein interactions between SNARE complexes by forming an ionic couple. Accordingly, we generated transgenic Drosophila lines expressing SNAREs mutated in these residues and performed an electrophysiological analysis of their neuromuscular junctions. Our results indicate that SNAP-25-R206 and syntaxin-D253 play a major role in neuroexocytosis and support a radial assembly of several SNARE complexes interacting via the ionic couple formed by these two residues.

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“…The resultant product was purified by agarose gel electrophoresis and cloned in pCR 2.1 vector. The cloned L chain gene was verified by DNA sequencing and subcloned in pGEX-4T2 vector containing the GST coding sequence (17).…”

Section: Methodsmentioning

confidence: 99%

Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A

Rigoni

Caccin

Johnson

et al. 2001

Biochemical and Biophysical Research Communications

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Recombinant and Truncated Tetanus Neurotoxin Light Chain: Cloning, Expression, Purification, and Proteolytic Activity

Cited by 15 publications

References 16 publications

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

Evidence for a radial SNARE super-complex mediating neurotransmitter release at the Drosophila neuromuscular junction

Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A

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