Inhibition of Vesicular Secretion in Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative of
            <i>Clostridium botulinum</i>
            Neurotoxin Type A

Chaddock, John A.; Purkiss, John R.; Friis, Lorna M.; Broadbridge, Janice D.; Duggan, Michael; Fooks, Sarah J.; Shone, Clifford C.; Quinn, Conrad P.; Foster, Keith A.

doi:10.1128/iai.68.5.2587-2593.2000

Cited by 61 publications

(43 citation statements)

References 28 publications

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“…It is also known that formation of the SNARE complex is a universal mechanism of vesicle fusion and secretion, not limited to neuronal cell types. To circumvent the limited availability of the requisite toxin receptor(s) on the target cell of interest, we have previously reported the replacement of the H C domain with a variety of ligands and retargeting of the LH N /A fragment into a range of neuronal and non-neuronal cells (9,13). In this study we have further developed the technology of retargeting clostridial endopeptidases with a view to endowing the conjugate with an ability to selectively target cells of potential therapeutic interest relative to anatomically and functionally closely related cells.…”

Section: The Clostridial Neurotoxin (Cnt)mentioning

confidence: 99%

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinumToxin A Endopeptidase Fragment and Erythrina cristagalliLectin

Duggan¹,

Quinn²,

Chaddock³

et al. 2002

Journal of Biological Chemistry

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108

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Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH N /A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH N /A or recombinant LH N /A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH N /A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved. The clostridial neurotoxin (CNT)1 family includes tetanus toxin (TeNT), produced by Clostridium tetani, and the seven antigenically distinct botulinum neurotoxins produced from strains of Clostridium botulinum (BoNTs). These proteins are responsible for the conditions of tetanus and botulism, respectively, that develop as a direct result of inhibition of Ca 2ϩ -dependent neurotransmitter release, a mechanism of action common to all the CNTs. In the case of BoNTs, intoxication of the neuromuscular junction is thought to occur in at least three phases: an initial binding phase, an internalization phase, and finally a neurotransmitter blockade phase (1).All CNTs have a similar structure and consist of a heavy chain (ϳ100 kDa) covalently joined to a light chain (ϳ50 kDa) by a single disulfide bond. Proteolytic cleavage of the heavy chain of C. botulinum neurotoxin type A (BoNT/A) generates two fragments of ϳ50 kDa each. The C-terminal domain (H C ) is required for target cell binding, with the N-terminal domain (H N ) being proposed to be involved in intracellular membrane translocation (2). Under conditions in which the disulfide bond between the light and heavy chains is maintained, trypsin cleavage results in a 100-kDa species termed LH N /A (light chain plus N-terminal heavy chain domain) representing a catalytically active, non-cell binding, non-toxic derivative of BoNT/A. In addition to obtainin...

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Section: The Clostridial Neurotoxin (Cnt)mentioning

confidence: 99%

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinumToxin A Endopeptidase Fragment and Erythrina cristagalliLectin

Duggan¹,

Quinn²,

Chaddock³

et al. 2002

Journal of Biological Chemistry

Self Cite

108

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“…In addition, a reduced toxicity of BoNT/A in the absence of the H C domain was reported (57), seemingly refuting the substantial contribution of the H N domain in the toxin-cell entry mechanism. However, Chaddock et al (58) reported that the H N domain-L chain not only exhibited in vivo toxicity at high concentrations but also inhibited neurotransmitter release in embryonic spinal cord neurons.…”

Section: Discussionmentioning

confidence: 95%

The C-Terminal Heavy-Chain Domain of Botulinum Neurotoxin A Is Not the Only Site That Binds Neurons, as the N-Terminal Heavy-Chain Domain Also Plays a Very Active Role in Toxin-Cell Binding and Interactions

2015

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c Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (H C ) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy- Botulinum neurotoxins (BoNTs) are the most potent toxins and are ranked by the Centers for Disease Control and Prevention (CDC) as bioterrorism threats with the six highest-risk class A agents (1). The potency of the toxin is due to its outstanding specificity to nerve terminals and selective enzymatic activity, resulting in the cleavage of SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) complex proteins. The cleavage of SNARE proteins inhibits the fusion of synaptic vesicles (containing the neurotransmitter) to the plasma membrane, thereby blocking neurotransmitter release and causing paralysis (2, 3).Unraveling the features of the mode of action of BoNTs has permitted numerous applications of the toxins in many therapeutic, pharmaceutical, and cosmetic applications (4-7). However, understanding of the full biochemical mechanism of its action still remains to be elucidated.BoNTs, produced by Gram-positive, obligate anaerobic Clostridium botulinum bacteria, are classified into eight different serotypes (A through H) (8-10). BoNTs are expressed as a single polypeptide chain (ϳ150 kDa) which is posttranslationally cleaved to produce an N-terminal light (L) chain of ϳ50 kDa, which is a Zn 2ϩ metalloprotease, and a heavy (H) chain of ϳ100 kDa. The L and H chains are linked together by a disulfide bond and other noncovalent interactions to form the active toxin. The H chain is further functionally divided into two seemingly independent domains: the N-terminal or translocation (H N ) domain and the Cterminal receptor-binding (H C ) domain (11).The entry of BoNT into neurons and its resultant toxicity are exhibited in a systematic manner, accomplished by a superb molecular partnership between the H and L chains. It is believed that an intricate mechanism involving various low-and high-affinity interactions is involved in intoxication of the cell (12). The difference in pH and redox potential across the endosomes is believed to induce conformational changes in the H chain, which subsequently forms a chaperone and channels the L chain into the cytosol. Once the L chain is translocated, it is subsequently released by disulfide bond reduction, followed by its refolding in the neutral cytosol, where it cleaves the SNARE substrates (13). The current understanding of toxin entry into the cell is that the H C domain binds to the neurons via dual host receptors (gangliosides...

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“…After cell binding and internalization, the H N domain of the TSI enables translocation of a selected LC to the cytosol to specifically cleave a member of the SNARE protein family and ultimately durably inhibit secretion (26). Some in vitro reports have validated this strategy in PC12 cells (27), in HIT-T15 B cells (8), and in embryonic dorsal root ganglion neurons (28).…”

Section: Discussionmentioning

confidence: 99%

“…The applicability of BoNTs' pharmacological properties to nonneural cells, which enhances their therapeutic value (7), has been enabled by a new approach in which the original C-terminal domain of the HC is replaced by a ligand domain specific to a selected cell-type receptor, promising to deliver targeted secretion inhibitors (TSI). In this sense, the chemical conjugation of a BoNT derivative retaining catalytic activity with an alternative cell-binding ligand was previously shown to inhibit the secretory mechanisms in different cell types in vitro (8).…”

Section: Introductionmentioning

confidence: 99%

A botulinum toxin–derived targeted secretion inhibitor downregulates the GH/IGF1 axis

Somm

Bonnet²,

Martínez³

et al. 2012

J. Clin. Invest.

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. In vitro, SXN101742 targeted the GHRH receptor and depleted a SNARE protein involved in GH exocytosis, vesicle-associated membrane protein 2 (VAMP2). In vivo, administering SXN101742 to growing rats produced a dose-dependent inhibition of GH synthesis, storage, and secretion. Consequently, hepatic IGF1 production and resultant circulating IGF1 levels were reduced. Accordingly, body weight, body length, organ weight, and bone mass acquisition were all decreased, reflecting the biological impact of SXN101742 on the GH/IGF1 axis. An inactivating 2-amino acid substitution within the zinc coordination site of the endopeptidase domain completely abolished SXN101742 inhibitory actions on GH and IGF1. Thus, genetically reengineered BoNTs can be targeted to nonneural cells to selectively inhibit hormone secretion, representing a new approach to treating hormonal excess.

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Inhibition of Vesicular Secretion in Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative of Clostridium botulinum Neurotoxin Type A

Cited by 61 publications

References 28 publications

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinumToxin A Endopeptidase Fragment and Erythrina cristagalliLectin

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinumToxin A Endopeptidase Fragment and Erythrina cristagalliLectin

The C-Terminal Heavy-Chain Domain of Botulinum Neurotoxin A Is Not the Only Site That Binds Neurons, as the N-Terminal Heavy-Chain Domain Also Plays a Very Active Role in Toxin-Cell Binding and Interactions

A botulinum toxin–derived targeted secretion inhibitor downregulates the GH/IGF1 axis

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