Identification of an ion channel‐forming motif in the primary structure of tetanus and botulinum neurotoxins

Monta, Myrta S.; Blewitt, Richard; Tomich, John M.; Montal, Mauricio

doi:10.1016/0014-5793(92)81173-j

Cited by 72 publications

(35 citation statements)

References 39 publications

(34 reference statements)

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“…Molecular modeling, energy minimization, and molecular dynamics simulations, were conducted on a Silicon Graphics IRIS 4D/210 GTXB workstation using the INSIGHT and DIS-COVER molecular modeling packages of Biosym (San Diego, California) (Oiki et al, 1990;Montal et al, 1990Montal et al, , 1992. Lowenergy arrangements of a-helices and four-helix bundles were calculated with semiempirical potential energy functions and optimization routines.…”

Section: Con Formational Energy Computationsmentioning

confidence: 99%

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

et al. 1995

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Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel-forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation-selective channels when reconstituted in planar lipid bilayers. As predicted, the selfassembled conductive oligomers express heterogeneous single-channel conductances. The most frequent openings exhibit single-channel conductance of 12 and 7 pS in 0.5 M NaCI, and 29 and 9 pS in 0.5 M KC1. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic a-helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel-forming activity of intact botulinum toxin A.

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Section: Con Formational Energy Computationsmentioning

confidence: 99%

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

et al. 1995

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“…After binding, the toxin is internalized into an endosome through receptor-mediated endocyctosis [17,18]. The amino-terminal half of the heavy chain (H N ) is believed to participate in the translocation mechanism of the light chain across the endosomal membrane [4,[19][20][21][22]. The low pH environment of the endosome may trigger a conformational change in the translocation domain, thus forming a channel for the light chain.…”

Section: Introductionmentioning

confidence: 99%

Development of vaccines for prevention of botulism

2000

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-Botulism is a potentially lethal disease caused by one of seven homologous neurotoxic proteins usually produced by the bacterium, Clostridium botulinum. This neuromuscular disorder occurs through an exquisite series of molecular events, ultimately ending with the arrest of acetylcholine release and hence, flaccid paralysis. The development of vaccines that protect against botulism dates back to the 1940s. Currently, a pentavalent vaccine that protects against BoNT serotypes A-E and a separate monovalent vaccine that protects against BoNT serotype F are available as Investigational New Drugs. However, due to the numerous shortcomings associated with the toxoid vaccines, several groups have efforts towards developing next-generation vaccines. Identifying a synthetic peptide that harbors a neutralizing epitope is one approach to a BoNT vaccine, while another employs the use of a Venezuelan equine encephalitis virus replicon vector to produce protective antigens in vivo against BoNT. The strategy used in our laboratory is to design synthetic genes encoding non-toxic, carboxy-terminal fragments of the C. botulinum neurotoxins (rBoNT(H C )). The gene products are expressed in the yeast, Pichia pastoris, and purified to greater than 98% with yields typically ranging from 200-500 mg per kg of wet cells. Protective immunity to the purified products against high-level challenges of neurotoxin is elicited in mice and in non-human primates. A pre-Investigational New Drug meeting was held with the Food and Drug Administration, and the next milestone for the vaccine candidates will be clinical trials. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS botulinum neurotoxin / vaccine / C-fragment / purification / efficacy

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“…Native gel electrophoresis and chemical crosslinking experiments have shown that TeNT forms dimers and trimers in solution [26]. Previous cryoelectron microscopy [23] and ion conductance [24] studies have also shown that both botulinum toxin and TeNT can form tetrameric channels in neuronal membranes. However, higher order TetC oligomers, such as trimers or tetramers, were not observed in the ESI-MS data presented here, probably because of the mass range limitations of the Mariner mass spectrometer used.…”

Section: Tetc Dimerizationmentioning

confidence: 97%

“…Following its binding to gangliosides and a protein receptor, TeNT oligomerizes and is pulled into an endosome where it forms a channel or pore in the cell membrane through which it transfers its catalytic domain [22][23][24][25]. Native gel electrophoresis and chemical crosslinking experiments have shown that TeNT forms dimers and trimers in solution [26].…”

Section: Tetc Dimerizationmentioning

confidence: 99%

Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

Conway

Whittal

Baldwin

et al. 2006

J. Am. Soc. Mass Spectrom.

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The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (K D values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc) n oligomers (n ϭ 1-6). K D values were found to range between ϳ10 -35 M. The strength of the interactions between the C fragment and (NeuAc) n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells. (J Am Soc Mass Spectrom 2006, 17, 967-976)

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Identification of an ion channel‐forming motif in the primary structure of tetanus and botulinum neurotoxins

Cited by 72 publications

References 39 publications

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

Development of vaccines for prevention of botulism

Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

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