Snake Venom Neurotoxins Research Articles

Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. α-Bungarotoxin purified from this venom failed to block transmission at 50μg/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1–3μg/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from α-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by α-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [ 125I]toxin F bound to the shared site and had no effect on [ 125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.

Kappa-bungarotoxin: a probe for the neuronal nicotinic acetylcholine receptor

Snake venom α-neurotoxins are excellent ligands for identifying and characterizing nicotinic acetylcholine receptors from skeletal muscle and muscle derived tissues. An α-neurotoxin referred to as Kappa-bungarotoxin (KBgT) has recently been isolated. KBgT is among a class of α-neurotoxins which are unusual in that they block nicotinic transmission in a number of neuronal preparations where most other α-neurotoxins are ineffective. KBgT appears to be a specific probe for the neuronal nicotinic receptor.

A comparison of nerve transection and chronic application of β-bungarotoxin on acetylcholine receptor distribution and other nerve-muscle properties

β-Bungarotoxin (β-BuTX), a snake venom neurotoxin which acts presynaptically to inhibit acetylcholine (ACh) release at the neuromuscular junction, was applied to the rat phrenic nerve-diaphragm muscle preparation to determine its effectiveness to mimic denervation. The distribution of junctional and extrajunctional ACh receptors on the muscle were assayed biochemically by [ 125I]α-bungarotoxin ([ 125I]α-BuTX) binding and electrophysiologically by iontophoretic application of ACh. Spontaneous transmitter release and muscle membrane potential were measured under conditions of denervation, β-BuTX treatment, and bee venom phospholipase A 2 exposure. Within 7 days after treatment with a single dose (5μg/kg) of enzymatically active β-BuTX, extrajunctional [ 125I]α-BuTX binding increased fivefold, and there was a decrease in miniature end-plate potential (MEPP) frequency and in resting membrane potential (RMP) to values less than those of control muscles but greater than those of denervated muscles. The same dose of enzymatically inactive β-BuTX or snake venom phospholipase A 2 was without effect, but a fivefold greater dose of enzymatically inactive β-BuTX resulted in changes in extrajunctional binding and RMP similar to those of muscles exposed to the enzymatically active toxin. However, unlike muscles treated with active toxin, those treated with inactive toxin had MEPP frequencies similar to control muscles and exhibited contraction elicited by phrenic nerve stimulation. These results taken together indicate that the nerve exerts a trophic influence on muscle independent of ACh and muscle activity.

β-Bungarotoxin-induced cell-death of neurons in chick retina

The cytotoxicity of β-bungarotoxin (β-BTX), a snake venom neurotoxin with phospholipase A 2 activity, for chick neurons was investigated using organ and monolayer cultures of retina. β-BTX led to a marked reduction in the total activities of choline acetyltransferase and glutamate decar☐ylase of retina cultures at concentrations as low as 100 pM. The total activity of lactate dehydrogenase was, however, much less affected by β-BTX. Also, the total activity of tyrosine hydroxylase of organ-cultured retina decreased only at 30–50 fold higher concentrations of the toxin. The total activity of the glial marker glutamine synthetase was not changed by β-BTX. In contrast to this selectivity for neurons displayed by β-BTX, non-neurotoxic phospholipases A 2 from bee venom and porcine pancreas led to a simultaneous loss of both neuronal and glial marker enzymes. Light and electron microscopy of organ-cultured retina showed that only cells in the ganglion cell layer and the inner third of the amacrine cell layer degenerated after incubation with β-BTX. In the toxin-sensitive cells, the Golgi apparatus and the endoplasmatic reticulum appeared the first subcellular structures to be affected. It is concluded that β-BTX preferentially recognizes and/or destroys cholinergic and GABAergic cells in the amacrine and ganglion cell layers of the developing chick retina. This toxin may thus be a useful probe to investigate cell surface properties of cholinergic and GABAergic neurons in the chick central nervous system.

Specific binding to isolated acetylcholine receptor of a synthetic peptide duplicating the sequence of the presumed active center of a lethal toxin from snake venom.

To verify the existence of a lethal "active center" in snake venom neurotoxins and to assess its delineation within their polypeptide sequences, a tritriacontapeptide matching residues 16-48 of the natural "major" toxin of Naja naja philippinensis (Hauert, J., Maire, M., Sussmann, A., and Bargetzi, J. P. (1974) Int. J. Pept. Protein Research 6, 201-222) has been synthesized by solid-phase technology (Juillerat, M. A., and Bargetzi, J. P. (1980), results presented at the 16th European Peptide Symposium, Helsingor, Denmark, September, (1980). After deblocking, cyclization by reoxidation, and purification, one of the resulting peptides exhibiting the correct chemical and physical characteristics was found to be highly "active" in binding isolated, purified, and standardized acetylcholine receptor protein. A new assay procedure had been developed using 3H-labeled alpha-bungarotoxin as nonreversible back-titrant. It has the advantage of measuring only specific binding of an unknown ligand competing for the same receptor protein. The observed KD was 2.2 x 10(-7) M, a value attesting to a higher affinity than acetylcholine itself, 2.5 x 10(-6) M, as well as curare and the small organic cholinergic ligands, albeit somewhat lower than the affinity of the parent native toxin, as expected from differences in molecular size.

Notexin preferentially inhibits the release of newly synthesized acetylcholine from rat brain synaptosomal fractions

An investigation was made of the effects of the snake venom neurotoxin, notexin, on acetylcholine turnover in rat brain P2 fractions using a gas chromatographic mass spectrometric assay for acetylcholine and choline. In contrast to earlier reports, we found a stimulation of the uptake and acetylation of labeled choline by toxin-treated P2 fractions. More significantly, notexin inhibited the release of this newly synthesized transmitter. These effects were found to be dependent on the dose of the toxin and the time of exposure of the P2 fraction to notexin. Longer exposure to notexin or experiments involving resuspension of notexin-treated P2 fractions appeared to result in considerable lysis of the transmitter-containing particles. Thus, notexin may alter acetylcholine compartmentation in the nerve ending and thereby affect acetylcholine synthesis.

Effects of post- and pre-synaptic snake venom neurotoxins on chloride and choline association with synaptic plasma membranes in vitro

The association of chloride-36 and [ 3H]choline with an isolated presynaptic plasma membrane fraction (SPM) was investigated. Choline interaction at low trans choline (1 μM) concentration was hemicholinium-3 sensitive and inhibited by the presynaptically active snake neurotoxins, taipoxin, Notechis II–5, Enhydrina schistosa myotoxin and β-bungarotoxin while the post-synaptic toxin α-bungarotoxin had no effect on choline interaction. Chloride interaction was not affected significantly by the toxins, thereby suggesting that gross lysis of the SPM does not account for their inhibitory effects on choline interaction with the SPM.

Composition of acetylcholine receptor protein from skeletal muscle.

THE isolated acetylcholine-recognition unit of the receptor protein (AChR) is characterised by its specific binding of snake venom α-neurotoxins and of nicotinic cholinergic ligands1–4. AChR purified completely from mammalian denervated muscle consists, chiefly or entirely, of an oligomer of 41,000 molecular weight subunits. It is not clear whether this comprises the entire receptor system, or whether another component is involved5,6 in forming the ionic channel.

Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms

Several snake venom neurotoxins are larger and more complex than the well-studied group of postsynaptic toxins exemplified by alpha-bungarotoxin. Several of these, exemplified by beta-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of detergents. The high hemolytic activity of crotoxin, the neurotoxin of Crotalus durissus terrificus, in the presence of lecithin has been attributed to this activity. The phospholipase A2 activity of several snake venom proteins has now been compared under the physiological conditions of the hemolysis tests. It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by component A under these conditions, or in the presence of deoxycholate. Phosphatidylserine is found to be digested more readily than egg white phosphatidylcholine; and also causes hemolysis in conjunction with much lower levels of crotoxin. In neither case is calcium required or stimulating. Phospholipase from Crotalus adamanteus, which is not neurotoxic, digests phosphatidylcholine more rapidly than does crotoxin, but phosphatidylserine more slowly; yet it is slightly less active than crotoxin in the hemolysis test with phosphatidylcholine, and much less with phosphatidylserine. The digestion of several phospholipids by either enzyme fails to release the expected protons in the absence of detergents at 37 degrees .beta-Bungarotoxin, highly neurotoxic, has negligible phospholipase A2 activity in the absence of detergents, and is almost nonhemolytic in conjunction with all phospholipids tested.Binding studies with (125)I-labeled compounds show that rabbit erythrocytes and ghosts have much greater affinity for crotoxin than for beta-bungarotoxin and do not bind Crotalus adamanteus phospholipase. The crotoxin complex is split in the course of binding, with only component B, the hemolytic component, becoming bound. It appears that the role of component A may be to diminish the nonspecific binding tendency of component B. Our data appear to be consistent with the concepts that affinity to membranes, particularly to specific sites on synaptic membranes, is the critical requirement for beta type neurotoxicity, and that this property, at least in some instances, has evolved from phospholipase A2 enzymes, but does not necessarily require retention and expression of enzymatic activity.

Taipoxin, an extremely potent presynaptic snake venom neurotoxin Elucidation of the primary structure of the acidic carbohydrate-containing taipoxin-subunit, a prophospholipase homolog

FEBS LettersVolume 84, Issue 2 p. 367-371 Full-length articleFree Access Taipoxin, an extremely potent presynaptic snake venom neurotoxin Elucidation of the primary structure of the acidic carbohydrate-containing taipoxin-subunit, a prophospholipase homolog J. Fohlman, J. Fohlman Institute of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, SwedenSearch for more papers by this authorP. Lind, P. Lind Institute of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, SwedenSearch for more papers by this authorD. Eaker, D. Eaker Institute of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, SwedenSearch for more papers by this author J. Fohlman, J. Fohlman Institute of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, SwedenSearch for more papers by this authorP. Lind, P. Lind Institute of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, SwedenSearch for more papers by this authorD. Eaker, D. Eaker Institute of Biochemistry, University of Uppsala, Box 576, S-751 23 Uppsala, SwedenSearch for more papers by this author First published: December 15, 1977 https://doi.org/10.1016/0014-5793(77)80726-6Citations: 55AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat References 1 J. Fohlman, D. Eaker, E. Karlsson, S. Thesleff, Eur. J. Biochem., 68, (1976), 457– 469. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 2 M. Kamenskaya, S. Thesleff, Acta Physiol. Scand., 90, (1974), 716– 724. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 3 S.G. Cull-Candy, J. Fohlman, D. Gustavsson, R. Lüllmann-Rauch, S. Thesleff, Neuroscience, 1, (1976), 175– 180. CrossrefCASPubMedWeb of Science®Google Scholar 4 M.J. Dowdall, J. Fohlman, D. Eaker, Nature, (1977), in press PubMedWeb of Science®Google Scholar 5 M. Belew, J. Fohlman, J.-C. Janson, Separation News (1977), Pharmacia in press Google Scholar 6 M. Belew, J. Porath, J. Fohlman, J.-C. Janson, J. Chromatogr., (1977), in press Web of Science®Google Scholar 7 E. Gross, B. Witkop, J. Biol. Chem., 237, (1962), 1856– 1862. CrossrefCASPubMedWeb of Science®Google Scholar 8 M. Koida, R. Walter, J. Biol. Chem., 251, (1976), 7593– 7599. CASPubMedWeb of Science®Google Scholar 9 B.M. Austen, E.L. Smith, Biochem. Biophys. Res. Commun., 72, (1976), 411– 417. CrossrefCASPubMedWeb of Science®Google Scholar 10 F. Joubert, Biochim. Biophys. Acta, 379, (1975), 345– 359. CrossrefCASPubMedWeb of Science®Google Scholar 11 F. Joubert, Eur. J. Biochem., 52, (1975), 539– 544. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 12 F. Joubert, Biochim. Biophys. Acta, 493, (1977), 216– 227. CrossrefCASPubMedWeb of Science®Google Scholar 13 Obidairo, T.K., Tampitag, S. and Eaker, D. (1977) to be published. Google Scholar 14 J. Halpert, D. Eaker, J. Biol. Chem., 250, (1975), 6990– 6997. CASPubMedWeb of Science®Google Scholar 15 J. Halpert, D. Eaker, J. Biol. Chem., 251, (1976), 7343– 7347. CASPubMedWeb of Science®Google Scholar 16 J.J. Volwerk, W.A. Pieterson, Haas G.H. De, Biochemistry, 13, (1974), 1446– 1454. CrossrefCASPubMedWeb of Science®Google Scholar 17 J. Drenth, C.M. Enzing, K.H. Kalk, J.C.A. Vessies, Nature, 264, (1976), 373– 377. CrossrefCASPubMedWeb of Science®Google Scholar 18 J. Fohlman, D. Eaker, M.J. Dowdall, Eur. J. Biochem., (1977), submitted Google Scholar 19 A. Evenberg, H. Meyer, W. Gaastra, H.M. Verheij, Haas G.H. De, J. Biol. Chem., 252, (1977), 1189– 1196. CASPubMedWeb of Science®Google Scholar 20 J. Halpert, D. Eaker, J. Biol. Chem., 250, (1975), 6990– 6997. CASPubMedWeb of Science®Google Scholar Citing Literature Volume84, Issue2December 15, 1977Pages 367-371 ReferencesRelatedInformation

Inhibition of high-affinity choline transport in peripheral cholinergic endings by presynaptic snake venom neurotoxins.

USE of purified snake venom neurotoxins with potent curarimimetic action on peripheral cholinergic transmission (for example, α-bungarotoxin1) in the study of the biochemical properties of the nicotinic cholinoceptor has met with considerable success (see ref. 2 for review). Some of the crude venoms from which such toxins have been isolated also contain toxins which act presynaptically, such as β-bungarotoxin1, notexin3 and taipoxin4. These three toxins inhibit the release of acetylcholine by a mechanism which is independent of the release process per se3–7. We show here that in an in vitro membrane preparation8 derived from the cholinergic endings in Torpedo electric organ these and related toxins are potent inhibitors of the high-affinity choline transport system. These observations provide a biochemical explanation for the inhibitory action of these toxins on transmitter release as they effectively block re-synthesis of acetylcholine from choline.

Molecular Graphics: Application to the Structure Determination of a Snake Venom Neurotoxin

Atomic coordinates have been determined for a snake venom alpha-neurotoxic protein by fitting a molecular model to a crystallographically derived 2.2-angstrom electron density map. The fitting was carried out entirely on a computer-operated molecular, graphics system without going through any mechanical model stage.

The crystal structure of a post-synaptic neurotoxin from sea snake at 2.2 Å resolution

Among the most lethal proteins in the venoms of sea snakes and elapid land snakes are the post-synaptic neurotoxins. These act by blocking the nicotinic acetylcholine receptor of the muscle motor end plate [ 1 ] , thus preventing transmission across the cholinergic synapse. Because of their extremely specific and tight binding to the receptor (KD is on the order of lo-“), these toxins have been the object of intensive chemical study [2], and have also been used to isolate the receptor protein [3,4] . Up to now there has been no information on the three-dimensional structure of the neurotoxins to aid in understanding their mode of action. We have solved the crystal structure of a neurotoxin from the sea snake Laticauda semifasciata from the Philippines Sea. This is a ‘short’ neurotoxin of 62 amino acids cross-linked by four disulfide bridges [S] . The structure has been determined at 2.2 A resolution. The molecule is a disc with one extended loop containing most of the residues believed essential for toxicity. We conclude that this structure is common to all snake venom neurotoxins, and that the postsynaptic toxins act by inserting the loop into a cleft or channel in the acetylcholine receptor.

Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor.

The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map. Erabutoxin b is one of a family of snake venom neurotoxins, all low-molecular-weight proteins, which block neuromuscular transmission at the postsynaptic membrane. They specifically inhibit the acetylcholine receptor. The molecular shape is that of a shallow elongated saucer with a footed stand formed by the six-membered ring at the COOH-terminal end. The central core of the molecule is an assembly of four disulfide bridges. Three long chain loops emerge as broad fronds from the core region. Approximately 40% of the main chain is organized into a twisted antiparallel beta-pleated sheet of five short strands. In 28 snake venom neurotoxins of established sequence which inhibit the acetylcholine receptor, the four disulfide bridges and seven other residues remain invariant. Three substitution positions conserve residue type. In one wing of the molecule, there is a broad shallow depression which may characterize the reactive site. It is populated by the sevent invariant residues and two of the three type conserved residues. This region is "anchored" on the undersurface of the molecule by the hydroxyl group of Ser-9, the remaining conservatively substituted residue.

Studies of the blocking action of snake venom neurotoxins at receptor sites to putative central nervous system transmitters: Parmentier, J. and Carpenter, D. O., Armed Forces Radiobiology Research Institute, Bethesda, Maryland, U.S.A.

Studies of the blocking action of snake venom neurotoxins at receptor sites to putative central nervous system transmitters: Parmentier, J. and Carpenter, D. O., Armed Forces Radiobiology Research Institute, Bethesda, Maryland, U.S.A.

Biochemical and physiological properties of a purified snake venom neurotoxin which acts presynaptically.

AbstractA highly toxic protein has been isolated from the venom of the snake Bungarus multicinctus. No major contaminants were detected on isoelectric focusing, gel filtration, or gel electrophoresis. The molecular weight calculated from its amino acid composition (21,800) agreed well with that calculated from gel filtration and equilibrium sedimentation data. Gel electrophoresis showed that the toxin consisted of two subunits of molecular weight 8,800 and 12,400 held together by disulfide bonds. Both the structure, determined by circular dichroism, and the biological activity, determined by toxicity assay, were remarkably resistant to heating.In the rat phrenic nerve‐diaphragm preparation, the toxin caused a failure of neuromuscular transmission, after which subthreshold endplate potentials could be detected. The frequency of spontaneous miniature potentials was altered only to a very slight extent. The toxin appeared to have no postsynaptic activity. Similarities between the properties of this neurotoxin and that described by Lee and Chang (1966) suggest that it is identical to β‐bungarotoxin.

Snake venom toxins: The amino acid sequences of two toxins from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom

Two toxins, which may be neurotoxins, have been isolated from the venom of Jameson's mamba ( Dendroaspis jamesoni kaimosae) and their amino acid sequences determined. Toxin V n II ∗ is a 60-residue protein, devoid of methionine and phenylalanine. Its four histidinyl residues per molecule are the highest number noted so far among short snake venom neurotoxins. Toxin V N IIII is another 72-residue toxin that contains two tryptophanyl residues. It is devoid of histidine and methionine. Both toxins show a high degree of homology with similar toxins from D. polylepis as well as toxins from other genera. An atypical chymotryptic split which occurs at an invariant lysine residue in snake venom toxins, is found, and factors that may be responsible for this, are discussed.

A model of the three-dimensional structure of snake venom neurotoxins based on chemical evidence.

Data, from studies of covalent structure and chemical and physico‐chemical behaviour of 27 different homologous proteins with either neurotoxic, cardiotoxic or fytic activity isolated from the venoms of elapid or hydrophid snakes have been collected from the literature. Because of their small size (60–74 amino acid residues) and their high content of disulphide bridges (4 or 5 bridges) these proteins provide an unusually favourable opportunity for trials to construct models of three‐dimensional structure from indirect chemical evidence. Such a model has been built from atomic skeleton model units for the Naja nigricollis a‐toxin, which contains 61 amino acid residues and 4 disulfide bridges.The dizerences in amino acid sequences bet ween the proteins with neurotoxic and those with cardiotoxic or lytic activity have been used to assign certain residues as important for the general folding of the molecule and others as important for neurotoxic activity. The last‐mentioned residues are presumed to form the site that binds to the acetyl‐choline receptor.

Snake venom toxins. Structure-function relationships and phylogenetics.

Abstract 1. 1. A phylogenetic relationship was established for fifteen neurotoxins and one cardiotoxin from snake venoms. The genera Naja , Hemachatus , Dendroaspis , Bungarus and Laticauda are represented. 2. 2. Ancestor amino acid sequences were obtained for the nodes of this phylogenetic tree—leading to the postulate that a cardiotoxin-like structure was the ancient form of snake toxins and that the two sizes of neurotoxins evolved from this form. 3. 3. A review of published work on the structures of snake venom toxins leads to the conclusion that three regions of the snake venom neurotoxins are implicated in their physiological activity.

Enhancement of immunogenicity of snake venom neurotoxins

Enhancement of immunogenicity of snake venom neurotoxins