Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor

Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha-subunit peptide most likely contains all of the determinants for binding of the toxin and glycoprotein peptides present on the alpha-subunit, because these peptides bind to the 32-residue alpha-subunit peptide with the same or greater affinity as to the intact subunit.     

Purification and characterization of a neurotoxin from the venom of Ophiophagus hannah (king cobra)

A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins.     

Alpha-bungarotoxin binding to acetylcholine receptor membranes studied by low angle X-ray diffraction

The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. alpha-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-A resolution for Torpedo californica nAChR membranes in the absence and presence of alpha-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-A lattice spacing between stacked nAChR membrane pairs. In the presence of alpha-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 A, and this distance increased to 85 A in the presence of toxin. Electron density profile structures were calculated in the absence and presence of alpha-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, alpha-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer.     

Isolation, complete amino acid sequence and characterization of a previously unreported post-synaptic neurotoxin - AlphaN3, from the venom of Bungarus candidus

The Malayan krait (Bungarus candidus) is one of the medically most important snake species in Southeast Asia. The venom from this snake has been shown to posses both presynaptic and post-synaptic neurotoxins. We have isolated a previously uncharacterized post-synaptic neurotoxin - alphaN3 from the venom of B. candidus. Isolation of the toxin was achieved in three successive chromatography steps - gel filtration on a Sephadex G75 column, followed by ion exchange chromatography (Mono-S strong cationic exchanger) and a final reverse-phase chromatography step (PRO-RPC C18 column). Purified toxin alphaN3 was shown to have an apparent molecular weight of ∼7 to 8 kDa on SDS-PAGE. The complete amino acid sequence of toxin alphaN3 was determined by Edman degradation and was found to share a high degree of homology with known post-synaptic neurotoxins (93% with alpha-bungarotoxin from Bungarus multicinctus, 50% with alpha cobratoxin from Naja kaouthia). The intravenous LD₅₀ of toxin alphaN3 was determined to be 0.16 ± 0.09 μg/g in mice which is comparable to alpha-bungarotoxin from B. multicinctus. Experiments with isolated nerve-muscle preparations suggested that toxin alphaN3 was a post-synaptic neurotoxin that produced complete blockade of neuromuscular transmission by binding to nicotinic acetylcholine receptors.     

kappa-Bungarotoxin: complete amino acid sequence of a neuronal nicotinic receptor probe

The complete amino acid sequence of kappa-bungarotoxin, a neurotoxin isolated from the venom of the banded krait Bungarus multicinctus, has been determined by automated Edman analyses of the intact protein and peptides derived from digests with trypsin and chymotrypsin. kappa-Bungarotoxin consists of a single polypeptide chain of 66 amino acids with a molecular weight of 7313. It contains 10 cysteinyl residues, presumably arranged in 5 disulfide bonds, and is completely devoid of methionine and tryptophan. The amino acid sequence of kappa-bungarotoxin shows greatest homology to the curaremimetic postsynaptic long neurotoxins of which alpha-bungarotoxin is also a member. However, there are some striking differences between kappa-bungarotoxin and other members of this group which may explain its unusual ability to block neuronal acetylcholine receptors.     

Role of C-terminal tail of long neurotoxins from snake venoms in molecular conformation and acetylcholine receptor binding: proton nuclear magnetic resonance and competition binding studies

The role of the "C-terminal tail" segment of long neurotoxins has been investigated. The C-terminal four to five residues of alpha-bungarotoxin and Laticauda colubrina b have been cleaved off by carboxypeptidase P. The effect of such deletion on the toxin conformation has been monitored in proton nuclear magnetic resonance spectra and circular dichroism spectra. The removal of the C-terminal residues primarily affects the chemical shifts of proton resonances of the residues close to the cleavage site and does not induce a major conformational change. Therefore, the C-terminal tail of long neurotoxins does not appear to be important in maintaining the specific polypeptide chain folding. On the other hand, competition binding with tritium-labeled toxin alpha to Narke japonica acetylcholine receptor has revealed that cleavage of the C-terminal residues reduces the binding activity of alpha-bungarotoxin or Laticauda colubrina b to acetylcholine receptor. Thus it is likely that (the basic amino acid residues in) the C-terminal tail is directly involved in the binding of long neurotoxins to electric organ (and muscle) acetylcholine receptor.     

Amino-acid sequence of the minor neurotoxin from Acalyptophis peronii venom

Sea snakes, Acalyptophis peronii, were captured in the Gulf of Thailand and their venom was isolated. A. peronii venom contains two neurotoxins called major and minor toxin. The complete amino-acid sequence of the minor toxin was identified and compared to that of the major toxin. The only difference between the major and the minor toxins is in the 43rd residue. The major toxin at this position contains glutamine, while the minor toxin contains glutamic acid. The LD50 of the minor toxin is 0.170 microgram/g in mice when injected intravenously. The toxicity is slightly lower than that of the major toxins, which has an LD50 of 0.125 microgram/g.     

Toxic components of the venom of the Central Asian scorpion, Orthochirus scrobiculosus

Four polypeptide neurotoxins, possessing paralytic activity for mice, were isolated from the venom of the Central Asian black scorpion Orthochirus scrobiculosus. All these toxins, Os-1 - Os-4, were shown to be homogeneous by disc-electrophoresis and N-terminal group analyses. The amino acid composition of the toxins was determined, methionine residues being found in toxin Os-1. The neurotoxin Os-3 was subjected to tryptic and chymotryptic hydrolyses and its total amino acid sequence was established. It was shown that neurotoxin Os-3 consists of 67 amino acid residues with four intramolecular disulfide bonds.     

Amino acid sequence of a long-chain neurotoxin homologue, Pa ID, from the venom of an Australian elapid snake, Pseudechis australis

Pa ID, a long-chain neurotoxin homologue, was isolated from the venom of an Australian elapid snake, Pseudechis australis, and its amino acid sequence was determined by conventional methods. Pa ID was an acidic protein (pI = 6.2) and consisted of 68 amino acid residues. It did not show binding activity to the acetylcholine receptor of an electric ray (Narke japonica) nor lethal effect on mice, though the amino acid sequence is homologous with those of long-chain neurotoxins isolated from other elapid snakes (homology, 39-51%). In the sequence of Pa ID, a structurally invariant residue (Tyr-22) and two functionally invariant residues (Val/Ala-49 and Lys/Arg-50) in snake venom neurotoxins are replaced by a cysteine, an arginine, and a methionine residue, respectively, and furthermore, four common residues in long-chain neurotoxins, Gly-17, Ala-43, Ser-59, and Phe/His-66 are replaced by a glutamic acid, a threonine, a threonine, and a valine residue, respectively. The conformational change of the protein molecule caused by these replacements and the removal of a positive charge at position 50 are probably the reasons why Pa ID has lost the lethality.     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors.

Two novel alpha-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main alpha-conotoxin contained in the venom, whereas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 microM which can be compared to the IC50 of 0.31 microM found for the previously characterized alpha-MI isolated from the piscivorous Conus magus. The synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for alpha-GI and alpha-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 degrees C.     

Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12

Studies were conducted on the properties of 125I-labeled alpha-bungarotoxin binding sites on cellular membrane fragments derived from the PC12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.     

The mechanism for acetylcholine receptor inhibition by alpha-neurotoxins and species-specific resistance to alpha-bungarotoxin revealed by NMR

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.     

Characterization of the binding of alpha-bungarotoxin to bacterially expressed cholinergic binding sites

Bacterially expressed cDNA fragments of the alpha-subunit of the nicotinic acetylcholine receptor previously have been shown to bind alpha-bungarotoxin (Gershoni, J. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4318-4321). Here, a novel system has been developed in which totally synthetic alpha-bungarotoxin binding sites are expressed in Escherichia coli transformants. The amino acid sequences, alpha 184-200 and alpha 184-196 of the Torpedo californica alpha-subunit of the nicotinic acetylcholine receptor were expressed as trpE fusion proteins via the expression vector pATH2 and a method for the enrichment of these fusion proteins is described. Quantitative analysis of toxin binding to the recombinant binding sites demonstrates that they bind toxin with affinities of KD = 2.5 X 10(-7) and 4.7 X 10(-6) M, respectively. Furthermore, the pharmacological profile of alpha 184-200 qualitatively reflects that of the intact receptor. These data not only indicate that the area of alpha 184-200 is an essential element of the cholinergic binding site but that residues alpha 197-200 contribute a point of contact between the receptor and alpha-bungarotoxin.     

Binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the alpha subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate

In order to investigate structure-function relationships of a segment of the acetylcholine receptor alpha subunit, binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of Torpedo, calf, and human alpha subunits was compared using a solid-phase radioassay. The affinities of 125I-alpha-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of alpha-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies against an alpha-bungarotoxin-binding peptide of the alpha-subunit of the acetylcholine receptor

Polyclonal and monoclonal antibodies were raised against a peptide comprising residues 173-204 of the alpha-subunit of the acetylcholine receptor. The polyclonal and pooled monoclonal antibodies inhibited up to 50% of 125I-alpha-bungarotoxin binding to peptide 173-204. Some of the antibodies recognized native receptor but did not significantly affect alpha-bungarotoxin binding. Epitope mapping revealed that the antibodies are directed against residues 183-194 indicating this region is a major determinant of toxin binding. This region is most likely conformationally constrained in the native receptor.     

Recognition of the acetylcholine receptor binding site of a long-chain neurotoxin by toxin-specific monoclonal antibodies.

The present paper reports the preparation and characterization of two neutralizing monoclonal antibodies (Mabs), called MST1 and MST2, which bind at the central loop of a long-chain neurotoxin from cobra venom. The central loop is a critical region for the binding of the toxin to the nicotinic acetylcholine receptor. Some of the residues incorporated in the epitopes recognized by MST1 and MST2 have been identified on the basis of competition experiments using a set of 'chemical mutants' of the toxin. We show that MST1 and MST2 bind at the base and at the tip of the central loop of the toxin, respectively, however, only MST2 actually overlaps the acetylcholine receptor binding site. Accordingly, only MST2 is capable of recognizing all homologous toxins so far examined. MST2, therefore, mimicks, at least partially, the site by which the nicotinic acetylcholine receptor recognizes a long-chain neurotoxin.     

Purification and chemical characterization of the major neurotoxin from the venom of Pelamis plautrus.

A major toxin was isolated from the venom of the sea snake Pelamis platurus (yellow-bellied sea snake) by Sephadex G-50 and carboxymethylcellulose column chromatography. The LD50 of the pure toxin (Pelamis toxin a) was 0.044 mug/g in mice representing a tenfold increase in toxicity after purification. The toxin was homogeneous in acrylamide disc gel electrophoresis and eluted as a single peak after isoelectric focusing in a sucrose density gradient column. The isoelectric point was 9.69; thus it is a highly basic protein. The toxin contained 55 amino acid residues with four disulfide linkages. When all disulfide linkages were reduced and alkylated, the toxic action of the pure toxin disappeared leading to the conclusion that the disulfide bonds of the neurotoxin were essential for toxic action.     

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.     

"Weak toxin" from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [(125)I]alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm. In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N. kaouthia, representing short-type and long-type alpha-neurotoxins, respectively. WTX and alpha-cobratoxin displaced [(125)I]alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin. It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.