Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates

Bade, Steffen; Rummel, Andreas; Reisinger, Clemens; Karnath, Tino; Ahnert‐Hilger, Gudrun; Bigalke, Hans; Binz, Thomas

doi:10.1111/j.1471-4159.2004.02844.x

Cited by 97 publications

(81 citation statements)

References 60 publications

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“…Application of the elegant and sensitive measurement of single channels formed in membranes by BoNTs (33,34) should yield insights into how two linked LCs can be translocated via the pore-forming domain of the N-terminal half of HC A . Consistent with our observation, other studies have shown that BoNT D can translocate a completely unrelated enzyme fused to its LC, dihydrofolate reductase, albeit with a significant reduction in its catalytic activity (35).…”

Section: Fusion Of Lc E To Botim a Extends Its Duration Of Action Andsupporting

confidence: 80%

A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis

Wang

Zurawski

Meng

et al. 2011

Journal of Biological Chemistry

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Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT A ) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT A was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC A ) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6 -7 N terminus residues diminished BoNT A activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC E to a BoNT A enzymically inactive mutant (BoTIM A ) yielded a novel LC E -BoTIM A protein that targets neurons, and the BoTIM A moiety also delivers and stabilizes the inhibitory LC E , giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT E . LC E-BoTIM A (AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT E , reaffirming that these residues are critical for the persistent action of LC E -BoTIM A as well as BoNT A . LC EBoTIM A inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves. Botulinum neurotoxins (BoNTs)3 inhibit transmitter release from peripheral cholinergic neurons causing the lifethreatening flaccid paralysis underlying botulism (1). The most potent biological substances, their estimated lethal doses (LD 50 ) in humans are between 0.1 and 1 ng/kg. Seven BoNT serotypes (A-G), produced by Clostridium botulinum, are synthesized as pro-form single chain proteins (SC, M r ϳ150,000) and converted by either Clostridial or tissue proteases into fully active dichain (DC) forms, consisting of a protease domain (LC; M r ϳ50,000) linked to a heavy chain (HC; M r ϳ100,000) through disulfide and noncovalent bonds. BoNT A preferentially enters cholinergic nerve endings by binding via the C-terminal half of their HC to a membrane acceptor, a lumenal domain of synaptic vesicle protein 2 (2, 3). On the other hand, type E only binds the glycosylated synaptic vesicle protein 2 A/B isoforms (4), which are sparsely expressed on sensory neurons, explaining its lack of effects on trigeminal ganglionic neurons (TGNs) (5). These toxins undergo acceptor-mediated endocytosis (6, 7) with translocation of the LCs into the cytosol through a channel formed by the N-terminal half...

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Section: Fusion Of Lc E To Botim a Extends Its Duration Of Action Andsupporting

confidence: 80%

A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis

Wang

Zurawski

Meng

et al. 2011

Journal of Biological Chemistry

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“…6,19 These characteristics of BoNTs have been explored and successfully exploited for experimental transfer of cargo and enzymatically active proteins into neurons. 20,21 The suitability of BoNTs as vehicles for neuronal gene targeting and biomolecule delivery is further augmented by the existence of multiple serotypes (A-G), which have different ecto-acceptors for entry into various neuronal populations. 22,23 The pronounced specificity of BoNT/B for motor nerve endings and peripheral autonomic neurons renders it highly attractive as a potential targeting moiety.…”

Section: Introductionmentioning

confidence: 99%

Innocuous full-length botulinum neurotoxin targets and promotes the expression of lentiviral vectors in central and autonomic neurons

et al. 2011

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Fragments of botulinum neurotoxin (BoNT) have been explored as potential targeting moieties and carriers of biomolecules into neurons, although with lower binding and translocation efficiency compared with intact proteins. This study exploits a detoxified recombinant form of full-length BoNT/B (BoTIM/B) fused with core streptavidin (CS-BoTIM/B) for lentiviral targeting to central and autonomic neurons. CS-BoTIM/B underwent an activity-dependent entry into cultured spinal cord neurons. Coupling CS-BoTIM/B to biotinylated lentivirus-encoding green fluorescent protein (GFP) endowed considerable neuron selectivity to the vector as evident from the preferential expression of the reporter in neurons co-cultured with skeletal muscle cells. CS-BoTIM/ B-guided lentiviral transduction with the expression of a SNARE protein, SNAP-25 (S25), rendered non-susceptible to proteolysis by three BoNT serotypes, yielded a sizable decrease in cleaved S25 upon exposure of spinal cord neurons to these toxins. This was accompanied by synaptic transmission being spared from blockade by BoNT/A or BoNT/E, reflecting adequate translation and functional competence of recombinant multi-toxin-resistant S25. The augmented neurotropism conveyed on the lentivirus by CS-BoTIM/B was also demonstrated in vivo through enhanced expression of a reporter in intramural ganglionic neurons in the rat trachea, after injection of the targeted GFP-encoding lentivirus. Thus, a novel and realistic prospect for gene therapy of peripheral neuropathies is offered in this study through lentiviral targeting to neurons by CS-BoTIM/B.

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“…Furthermore, identification of both receptor sites provides an approach to retarget BoNTs to different cell types by site-directed mutagenesis. Such modified BoNTs could also be used as drug delivery systems (36).…”

Section: Interference Of Ganglioside and Syt Interaction Sites Of Bonmentioning

confidence: 99%

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

Rummel

Eichner

Weil

et al. 2007

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

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Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their HCC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.synaptotagmin ͉ tetanus B otulinum neurotoxins (BoNTs) (serotypes A-G) are the causative agents of the disease botulism. The neurotoxins are produced as Ϸ150-kDa single-chain proteins in Clostridium botulinum and subsequently cleaved by proteases, yielding an Ϸ100-kDa heavy chain (HC) and an Ϸ50-kDa light chain (LC). These chains remain connected via a single disulfide bridge, noncovalent interactions, and a HC-derived peptide loop wrapped around the LC. The LCs act as zinc metallopeptidases, which solely hydrolyze one of three SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins: vesicle associated membrane protein/synaptobrevin, synaptosomal-associated protein of 25 kDa, or syntaxin. Together, these substrate molecules constitute the core of the vesicular fusion machinery. Thus, cleavage of one of these proteins inhibits the release of neurotransmitters from synaptic vesicles into the synaptic cleft. The HCs mediate the neurospecific binding, uptake by receptor-mediated endocytosis, and transport of the LC across the endosomal membrane into the cytosol, where the LCs encounter their substrates. The Ϸ50-kDa

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Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates

Cited by 97 publications

References 60 publications

A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis

A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis

Innocuous full-length botulinum neurotoxin targets and promotes the expression of lentiviral vectors in central and autonomic neurons

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

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