Electric dipole reorientation in the interaction of botulinum neurotoxins with neuronal membranes

Botulinum neurotoxins are highly potent toxins capable of rapid and specific interaction with the presynaptic membrane. We have hypothesised that: (1) these neurotoxins possess an electric dipole with the positive pole on receptor binding domain Hc-C and that (2) on approaching the negatively charged presynaptic membrane, they reorient themselves and hit the membrane surface with Hc-C; this electrostatic effect would contribute efficient binding. Electrostatic calculations confirm these hypotheses and strongly indicate that electrostatics effects can play an important role in the unique presynaptic membrane binding properties of these neurotoxins and generally on the interaction of other plasma membrane protein ligands.     

Effect of pH on the interaction of botulinum neurotoxins A, B and E with liposomes.

The interaction of botulinum neurotoxin serotypes A, B and E with membranes of different lipid compositions was examined by photolabelling with two photoreactive phosphatidylcholine analogues that monitor the polar region and the hydrophobic core of the lipid bilayer. At neutral pH the neurotoxins interacted both with the polar head groups and with fatty acid chains of phospholipids. At acidic pHs the neurotoxins underwent structural changes characterized by a more extensive interaction with lipids. Both the heavy and light chain subunits of the neurotoxins were involved in the process. The change in the nature and extent of toxin-lipid interaction occurred in the pH range 4-6 and was not influenced by the presence of polysialogangliosides. The present data are in agreement with the idea that botulinum neurotoxins enter into nerve cells from a low pH intracellular compartment.     

Tetanus, botulinum and snake presynaptic neurotoxins

Tetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins known and are solely responsible for the pathogenesis of tetanus and botulism. They are metallo-proteases that enter nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.

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Mechanism of action of tetanus and botulinum neurotoxins

The clostridial neurotoxins responsible for tetanus and botulism are metallo-proteases that enter nerve cells and block neurotransmitter release via zinc-dependent cleavage of protein components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular Junction and is internalized and transported retroaxonally to the spinal cord. Whilst TeNT causes spastic paralysis by acting on the spinal inhibitory interneurons, the seven serotypes of botullnum neurotoxins (BoNT) induce a flaccid paralysis because they intoxicate the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G specifically cleave VAMP/synaptobrevin, a membrane protein of small synaptic vesicles, at different single peptide bonds. Proteins of the presynaptic membrane are specifically attacked by the other BoNTs: serotypes A and E cleave SNAP-25 at two different sites located within the carboxyl terminus, whereas the specific target of serotype C is syntaxin.     

肉毒神经毒素抑制剂的研究进展

肉毒神经毒素(botulinum neurotoxins,BoNTs)是由梭状芽孢杆菌属分泌的外毒素,是目前已知毒性最强的生物类毒素.BoNTs共分为7种血清型(A-G),其中A型导致的肉毒中毒最为常见.由于肉毒毒素的强毒性及易于制备,其已被列为A类生物恐怖制剂.目前,针对肉毒中毒的有效治疗手段为早期注射抗毒素血清.但...     

How botulinum and tetanus neurotoxins block neurotransmitter release

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.     

The journey of tetanus and botulinum neurotoxins in neurons

Anaerobic bacteria of the genus Clostridia are a major threat to human and animal health, being responsible for pathologies ranging from food poisoning to gas gangrene. In each of these, the production of sophisticated exotoxins is the main cause of disease. The most powerful clostridial toxins are tetanus and botulinum neurotoxins, the causative agents of tetanus and botulism. They are structurally organized into three domains endowed with distinct functions: high affinity binding to neurons, membrane translocation and specific cleavage of proteins controlling neuroexocytosis. Recent discoveries regarding the mechanism of membrane recruitment and sorting of these neurotoxins within neurons make them ideal tools to uncover essential aspects of neuronal physiology in health and disease.     

Unique ganglioside binding by botulinum neurotoxins C and D-SA

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G), based on a lack of cross-antiserum neutralization. The BoNT/C and BoNT/D serotypes include mosaic toxins that are organized as D-C and C-D toxins. One BoNT D-C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with a vaccine composed of either prototype BoNT/C-Stockholm or BoNT/D-1873. Whereas several BoNT serotypes utilize dual receptors (gangliosides and proteins) to bind to and enter neurons, the basis for BoNT/C and BoNT/D entry into neurons is less well understood. Recent studies solved the crystal structures of the receptor-binding domains of BoNT/C, BoNT/D, and BoNT/D-SA. Comparative structural analysis showed that BoNT/C, BoNT/D and BoNT/D-SA lacked components of the ganglioside-binding pocket that exists within other BoNT serotypes. With the use of structure-based alignments, biochemical analyses, and cell-binding approaches, BoNT/C and BoNT/D-SA have been shown to possess a unique ganglioside-binding domain, the ganglioside-binding loop. Defining how BoNTs enter host cells provides insights towards understanding the evolution and extending the potential therapeutic and immunological values of the BoNT serotypes.     

New equine antitoxins to botulinum neurotoxins serotypes A and B

Hyperimmune monovalent antitoxins to botulinum neurotoxin serotypes A and B have been produced by immunizing horses with newly developed formalin toxoids. After primary immunization, horses developed acceptable prophylactic antibody titers (1-5 IU/mL). Three horses received additional toxoid booster injections to induce hyperimmune antibody titers with antitoxin-A and antitoxin-B titers reaching peaks of approximately 2000 IU/mL and 150-625 IU/mL, respectively. Titers were quantified throughout the process by antigen-capture ELISA and by in-vivo neutralization. ELISA titers and neutralization titers correlated (R2 ～0.62-0.92), however, unique correlations between in-vitro and in-vivo titers were observed for each horse. Monovalent antitoxin pools were made by combining plasma that had been collected twice via plasmaphoresis several months after primary immunization. Neutralizing units were established for each pool relative to the current US and WHO reference standards. Titers were determined at the L(+)/10 and L(+)/40 toxin dose for Toxin types A and B, respectively, and U.S. and international units were assigned to each monovalent antitoxin. Avidity of the new Anti-A pool was equivalent to the WHO Anti-A reference at the L(+), L(+)/10 and L(+)/30 dose. Each monovalent plasma pool failed to cross-neutralize other botulinum neurotoxin serotypes indicating a high degree of specificity of each antitoxin for the toxin serotype used during immunization.     

Unique substrate recognition by botulinum neurotoxins serotypes A and E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.     

Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses

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

The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC-fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC-fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation of these HC-fragment/ganglioside complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full-length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl-terminal half of the HC-fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H...SXWY...G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.     

Partial amino acid sequences of botulinum neurotoxins types B and E

Clostridium botulinum type E neurotoxin, a single-chain protein of Mr 147,000, was purified and subjected to amino acid sequencing. The same was done for single-chain botulinum type B neurotoxin (Mr 152,000), and for the heavy and light chains (Mr 104,000 and 51,000 respectively) derived from type B by limited trypsin digestion. Twelve to eighteen residues were identified and the following conclusions were drawn: The light chain of the nicked (dichain) type B is derived from the N-terminal one-third of the single-chain (unnicked) parent neurotoxin; sequence homologies are present between single-chain types B and E and the light chain of the nicked type A [J. J. Schmidt, V. Sathyamoorthy, and B. R. DasGupta (1984) Biochem. Biophys. Res. Commun. 119, 900-904]; the N-terminal regions of the heavy chains of types A and B have some structural similarity; and activation of type B neurotoxin cannot involve removal of amino acids or peptides from the N terminus.     

Membrane interactions of tetanus and botulinum neurotoxins: a photolabelling study with photoactivatable phospholipids

Tetanus and botulinum neurotoxins (TeNT and BoNT) bind strongly and specifically to the nervous tissue, as it can be inferred from their potency and from their effects restricted to the nervous system. The molecular basis of these properties are presently unknown. As a first approach, we have investigated the interaction of TeNT and BoNT with model membranes by photolabelling with phospholipid analogues carrying the photoreceptor group at different positions of the lipid molecule in order to probe different membrane regions. We found that at neutral pH TeNT and BoNTs (type A, B and E) adsorb onto the surface of negatively charged liposomes. Polysialogangliosides increase this interaction only slightly thus suggesting that they provide a minor contribution to toxin lipid binding. On this basis we propose that clostridial neurotoxins bind to lipids via both a predominant unspecific interaction with negatively charged lipids (including gangliosides) and a specific, but weaker, interaction with polysialogangliosides. At acidic pH values both chains of these neurotoxins are labelled strongly by photogroups located in the hydrophobic milieu of the membrane with a pH dependence that overlaps the range of pH values reached in the endosomal lumen. This result is consistent with their insertion into the lipid bilayer in agreement with the idea that clostridial neurotoxins may penetrate into cells via intracellular low pH compartments.     

Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins

Until recently, all clostridia producing neurotoxins able to cause paralysis symptomatic of botulism were deemed to be Clostridium botulinum. Defining Cl. botulinum on the basis of this single phenotypic trait has resulted in the species encompassing metabolically very diverse organisms, and four distinct phenotypic groups are recognized within this taxon (designated groups I-IV). Nucleic acid hybridization and 16S ribosomal RNA sequencing studies have revealed the presence of four phylogenetically distinct lineages within the species, which correlate with these phenotypic divisions. In addition to marked phenotypic and genotypic heterogeneity between groups, the taxonomy of the species is further complicated by the existence of strains which are closely related, if not genetically identifiable, to members of each Cl. botulinum group, but are non-toxigenic. Furthermore, strains of species other than Cl. botulinum (viz. Cl. baratii, Cl. butyricum) have been found which express botulinum neurotoxin (BoNT). Great advances have been made in recent years in elucidating the nucleotide sequences of genes encoding the various BoNT antigenic types (A through to G). Genealogical trees derived from BoNTs show marked discordance with those depicting 'natural' relationships inferred from 16S rRNA and phenotypic clusters, and strong evidence exists for BoNT gene transfer between some groups of Cl. botulinum (e.g. groups I and II), and with non-botulinum species. Botulinum neurotoxin is produced by Cl. botulinum as a non-covalently bound progenitor toxin complex of two or more protein components. Information on the evolutionary histories of the various non-toxic progenitor proteins is currently limited, although there is evidence of gene recombination. In particular, chimera-like or mosaic non-toxic-non-haemagglutinins (NTNH) genes in group I Cl. botulinum have been described, and it is now apparent that the phylogeny of the NTNHs is not going to 'mirror' that of botulinal neurotoxins, although their genes are physically contiguous. In this article, the current state of knowledge of the phylogenetics of the species Cl. botulinum and its neurotoxins is reviewed, and a view is presented that a nomenclature based rigidly on BoNT production is no longer tenable.     

Enterotoxic properties of salmonellae and their neurotoxins

Most of live S. typhimurium cultures are capable of intraintestinal proliferation and possess enterotoxic activity. The capacity of S. typhimurium strains for producing enterotoxins is not connected with their origin. The parenteral immunization of rabbits with corpuscular vaccines prepared from S. typhimurium induced changes in the sensitivity of different sections of the small intestine of the animals to the enterotoxic action of live homologous cultures. Neurotoxin isolated from S. typhi was found to possess enterotoxic activity.     

不同血清型肉毒毒素受体结合域研究进展

肉毒毒素(botulinum neurotoxin, BoNT)是人类已知毒性最强的蛋白质之一,可以引起肌肉松弛麻痹,严重时可导致死亡。肉毒毒素共分为7种血清型(BoNT/A-BoNT/G),根据氨基酸序列差异可进一步分为40多种亚型。肉毒毒素分子结构由3个基本结构域组成：重链羧基端细胞受体结合域、氨基端的易位域和轻链催化域。在运动神经元表面,受体结合域首先与聚唾液酸神经节苷脂结合,随后与突触囊泡蛋白2或突触囊泡结合蛋白结合形成双受体复合物。每种血清型的受体结合域都必须与其相应受体结合才能发挥作用。肉毒毒素的结构功能及其对宿主的作用一直都是研究热点。近年来,因受体结合域可以促进肉毒毒素与运动神经元膜特异性结合,而成为新的研究方向。本综述将概述不同血清型肉毒毒素与受体结合过程中受体结合域结构变化和结合位点差异。通过分析不同血清型及亚型的序列以及受体结合域结构特征,可以更好地了解细胞受体结合域的序列差异和功能,并为肉毒毒素的治疗策略提供新思路。     

Role of metals in the biological activity of Clostridium botulinum neurotoxins

Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals. The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity. While the catalytic activity is a zinc endopeptidase activity on the SNARE complex proteins, the translocation is believed to be a pH-dependent process allowing the translocation domain to change its conformation to penetrate the endosomal membrane. Here, we report the crystal structures of botulinum neurotoxin type B at various pHs and of an apo form of the neurotoxin, and discuss the role of metal ions and the effect of pH variation in the biological activity. Except for the perturbation of a few side chains, the conformation of the catalytic domain is unchanged in the zinc-depleted apotoxin, suggesting that zinc's role is catalytic. We have also identified two calcium ions in the molecule and present biochemical evidence to show that they play a role in the translocation of the light chain through the membrane.