Short Neurotoxins Research Articles

Lipid-protein interactions. A comparative study of the binding of cardiotoxins and neurotoxins to phospholipid vesicles

It has recently been shown that cardiotoxin II from Naja mossambica mossambica specifically interacts with negatively charged phospholipids (Dufourcq, J. and Faucon, J.F. (1978) Biochemistry 17, 1170–1176). In order to investigate whether or not short neurotoxins give rise to similar interactions, four techniques have been used, namely intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene, turbidity measurements and release of 6-carboxyfluorescein trapped inside single shelled vesicles. Neurotoxin III from Naja mossambica mossambica and neurotoxin I from the venom of the scorpion Androctonus australis Hector, specifically interact with negatively charged phospholipids leading to changes in tryptophan fluorescence and to a decrease of the fluidity of the bilayer. Cardiotoxin II from the same snake venom gives similar results. On the other hand, it seems that either a very weak or no interaction at all occurs in the case of neurotoxin I from the same Naja venom. There are important differences in the behaviour of cardiotoxin and neurotoxins: (i) neurotoxins lead to only weak release of 6-carboxyfluorescein from lipid vesicles, whereas cardiotoxin II induces fast and quantitative escape of the dye and then a general breakdown of the vesicular structure; (ii) binding of neurotoxins can be easily reversed by 100–200 mM NaCl or less than 1 mM Ca 2+ and so it is essentially electrostatic, whereas binding of cardiotoxin II seems to involve some hydrophobic contribution. The short neurotoxins and cardiotoxins from snake venom having a great homology in sequence, their differences on binding properties are discussed in terms of changes in a particular area of the sequence.

Some properties and the complete primary structures of two reduced and S-carboxymethylated polypeptides (S 5C 1 and S 5C 10) from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom

Two polypeptides (protein S 5C 1 and toxin S 5C 10) were purified from Dendroaspis jamesoni kaimosae venom. Whereas protein S 5C 1 comprises 61 amino acid residues, toxin S 5C 10 contains 58 and they each comprise four disulphide bridges. The complete primary structures of the two polypeptides have been elucidated. The sequences of protein S 5C 1 and toxin S 5C 10 are structurally homologous to the short neurotoxins Type I, but they are much less toxic. In toxin S 5C 10 one of the functionally invariant amino acid residues, lysine 26, of the Type I neurotoxin has been replaced by a serine. In contrast protein S 5C 1 has the feature that it contains ten or eleven structurally invariant amino acids and apparently only one of the five functionally invariant residues.

Snake Venoms. The Amino-Acid Sequence of Protein S2C4fromDendroaspis jamesoni kaimosae(Jameson’s mamba) Venom

A major component (S2C4) was purified from Jameson's mamba by gel filtration on Sephadex G-50 and ion-exchange chromatography on CM-cellulose. Protein S2C4 comprises 62 amino acid residues including 8 half-cystine residues. The complete amino acid sequence of the protein has been established. The sequence and the invariant amino acid residues of protein S2C4 resemble a short neurotoxin, a long neurotoxin, a cytotoxin and an angusticeps type protein. However, the position of its four disulphide bridges differs from those encountered in a short neurotoxin or a cytotoxin. Mixtures of protein S2C4 and angusticeps type proteins revealed a marked synergistic effect, in that their toxicity in combination was greater than the sum of their individual toxicities.

Purification, some properties and the primary structures of three reduced and S-carboxymethylated toxins (CM-5, CM-6 and CM-10a) from Naje haje haje (egyptian cobra) venom

Three reduced and S-carboxymethylated toxins (CM-5, CM-6 and CM-10a) were purified from Naja haje haje (Egyptian cobra) venom. Whereas toxin CM-5 comprises 71 amino acid residues and five intrachain disulphide bridges, toxins CM-6 and CM-10a contain each 61 residues and four disulphide bridges. The complete primary structures of the three toxins have been established. The toxicity, the immunochemical properties, the sequence and the invariant amino acid residues of toxin CM-5 resemble the properties of the long neurotoxin group, while those of toxin CM-6 and CM-10a are related to the short neurotoxin group. Further, the sequences of the three toxins from Naja haje haje venom reveal a high degree of homology with those of the corresponding neurotoxins isolated from Naja haje annulifera or Naja nivea venoms.

Naja haje haje (Egyptian cobra) venom. Some properties and the complete primary structure of three toxins (CM-2, CM-11 and CM-12).

Three toxins (CM-2, CM-11 and CM-12) were purified from Naja haje haje (Egyptian cobra) venom. Whereas toxin CM-11 contains 65 amino acid residues and five intrachain disulphide bridges, toxin CM-2 and CM-12 comprise, respectively, 61 and 62 residues but both contain four disulphide bridges. The complete primary structures of the three toxins have been established. The sequence and the invarient amino acid residues of CM-2 resemble those of part of a long neurotoxin, a short neurotoxin and a cytotoxin. The sequence of CM-11 reveals that it is a homologue of the neurotoxins and to some extent also a cytotoxin. The immunochemical properties and the sequences of CM-12 suggest that it is related to the cytotoxin group. Further, the sequences of CM-11 and CM-12 from Naja haje haje venom show a high degree of homology with those of the corresponding toxins isolated from NaJA annulifera or NaJA melanoleuca venoms.

A possible structural basis for the different modes of action of neurotoxins and cardiotoxins from snake venoms

The short neurotoxins and the cardiotoxins from snake venoms are a large group of homologous proteins [ 1,2]. Spectroscopic studies indicated that the homology among these toxins includes also the threedimensional structure and that the conformation type found in single crystals of a particular neurotoxin, erabutoxin b [3,4], prevails quite generally for the proteins in this group [S-8]. On the other hand it appears that in spite of the structural similarities, the mechanism of action of the neurotoxins is quite different from that of the cardiotoxins. Neurotoxins bind to a protein receptor at the postsynaptic level and block acetylcholine transmission [9]. Cardiotoxins cause irreversible depolarization of celI membranes, e.g., desactivating the sodium-potassium adenosine triphosphatase [lo]. Evidence was presented that cardiotoxins bind to lipid components of cell membranes [lo], and the association of cardiotoxin with negatively charged phospholipids was demonstrated [ 11 ,I 21. We propose here a structural basis for the different functions of neurotoxins and cardiotoxins based on recent observations by others and in our laboratory. which are combined into an extended P-sheet comprising five antiparallel segments of the polypeptide chain. This is presented schematically in fig.1 A. (ii) From Raman scattering [5], proton NMR [6] and circular dichroism [8] it was concluded that the solution conformations of short neurotoxins and cardiotoxins contain comparable amounts of &structure, which correspond to that found in the crystal structure of erabutoxin b [3,4]. Additional ‘H NMR data show that certain nearest neighbor relations between amino acid side chains found in erabutoxin b are preserved in the solution conformations of other toxins [?,13]. Since it thus appears that the same structure type prevails for short neurotoxins and cardiotoxins, we have fitted the amino acid sequence of a cardiotoxin, cardiotoxin VJJ4 from Naja mossambica mossambica [ 141, to the backbone conformation of erabutoxin b. To accommodate the deletions of the residues 4,6 and 7 [ 141, the first hairpin loop was shortened and rearranged so that the locations of Leu 1, Lys 2 and the disulfide bond Cys 3-Cys 24 were preserved (fig. 1 B). A second smaller modification was made by the addition of a small bulge between the disultide bridges Cys 17-Cys 41 and Cys 4%Cys 56 to accommodate the insertion of the two residues Asn 43 and Va144. 2. proposed model for cardiotoxin-membrane interactions

The conformation of cardiotoxins and neurotoxins from snake venoms

The conformations of a number of snake venom cardiotoxins and short (60–62 residues) neurotoxins were compared with the aid of circular dichroism spectra, sequences and theoretical predictions. The near-ultraviolet CD spectra of cardiotoxins exhibited positive Cotton effects arising mainly from the side chain of an invariant tyrosine located at position 23 of their sequences. By contrast, neurotoxins without exception had negative near-ultraviolet CD extrema, even though they contain a tyrosine in an identical position. The reversal in CD sign was attributed to the unique presence of tryptophan-28 known to be involved in neurotoxicity and which presumably possesses the necessary negative ellipticity to dominate neurotoxin near-ultraviolet spectra. A prominent positive Cotton effect invariably observed near 230 nm in neurotoxin spectra was likewise assigned to this tryptophan. Despite these characteristic differences, the deep-ultraviolet CD spectra concerned with the conformation of the polypeptide backbone of the toxins were quite similar. All had a single negative extremum near 215 nm, which is indicative of β-conformation and the absence of helix. The comparative CD results and sequence-based theoretical predictions of structure indicated that the diverse biological effects of cardiotoxins and neurotoxins are not due to gross main chain conformational differences. Changes in local side chain structure within a larger common three-dimensional framework are postulated to be sufficient to confer different binding specificities on cardiotoxins and neurotoxins, resulting in their action on different target membranes.

Predicted secondary structure of snake venom toxins from their primary structures.

We have predicted the secondary structure of 38 snake venom toxins using the method of Chou and Fasman. Our predictions indicate that beta-chain and random coil structures predominate in these proteins. The conformations of long neurotoxins, short neurotoxins and cytotoxins are less similar than previously believed. Cytotoxins contain 40--50% of beta-structure and they form a notably homogeneous group. Short neurotoxins contain less beta-structure (13--30%) and more random coil than cytotoxins, and they also form a more heterogeneous group in terms of secondary structure. The characteristics of long neurotoxins are intermediate to the above mentioned groups. Experimental evidence supporting these propositions is discussed.

Molecular evolution of snake venom toxins.

Phylogenetic trees were constructed for 62 venom toxins of snakes of Proteroglyphae suborder using matrix method. The resulting tree from Minimum Spanning Tree-Cluster Analysis technique had the lowest "percent deviation" (8.55). The taxonomic relationship of these toxins agrees very well with zoological opinions. However, the appearance of the tree did not directly provide a plausible evolutionary model for the toxins. A model was derived from nodal ancestral sequence calculations, comparisons between intra- and intergenerical rates of amino acid change, and generally held ideas about protein evolution. According to the model, short neurotoxin is the ancient form of snake venom toxins. The courses of evolution leading to the present intraspecific homologous toxins are explained by gene duplication and allelomorphism.

Snake venom toxins. The amino-acid sequence of a short-neurotoxin homologue from Dendroaspis polylepis polylepis (black mamba) venom.

The third most abundant component of black mamba venom, named FS2, was sequenced with the aid of sequenator studies and peptides derived by tryptic and chymotryptic digestion. Cyanogen bromide digests provided extra information to support the proposed structure. This protein is a homologue of the short neurotoxins of snake venom, but is much less toxic. Its structure is quite different from both neurotoxins and the other mamba proteins, called angusticeps types (neurotoxin homologues). Comparison of the known angusticeps-type toxins from mamba venom with mamba neurotoxins and each other leads to proposals that these proteins of low toxicity are inventions of the group of mambas and that three different, as yet unknown, functions will be associated with the three subgroups that are discernable.

Snake venoms. The amino-acid sequence of polypeptide DE-1 from Ophiophagus hannah (King cobra) venom.

A major polypeptide (DE-1) was purified from King cobra venom by ion-exchange chromatography on CM-cellulose and also DEAE-cellulose and by gel filtration on Sephadex G-50. DE-1 comprises 60 amino acid residues and is cross-linked by four intrachain disulphide bridges. The complete primary structure of the polypeptide has been elucidated. The properties of DE-1 were compared with those of the short neurotoxin and the cytotoxin groups and of the angusticeps types. The sequence and some of the invariant residues of DE-1 resemble the short neurotoxin group and to some degree also the cytotoxin group, but it is a nontoxic (LD50 greater than 250 microgram/g of mouse) polypeptide.

Structure-function relationship in the binding of snake neurotoxins to the torpedo membrane receptor.

The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins associate 6-7 times faster with the toxin receptor and dissociate about 5-9 times faster that long neurotoxins. Acetylation and dansylation of Lys53 and Lys 27 decrease the affinity of long and short toxins for their receptor by a factor of about 200 at pH 7.4, 20 degrees, mainly because of the slower rate of association with the receptor.

Amino acid sequence of oxiana α, the main neurotoxin of the venom of Naja naja oxiana

The amino acid sequence of oxiana α, the main neurotoxin of Naja naja oxiana, has been determined. The molecule is a single chain of 61 amino acids cross-linked by four disulfide bridges and has a formula weight of 6786. It is homologous to other curarimimetic neurotoxins of the same size group (60–62 residues). A practical advantage of oxiana a among the short neurotoxins described so far is its comparatively easy availability. It accounts for 12% of the crude venom, which is about twice the level of most other prominant short neurotoxins in terrestrial snake venoms, and can be isolated directly from the crude venom in a single chromatographic step.

Snake Venom Toxins: THE PURIFICATION AND AMINO ACID SEQUENCE OF TOXIN TA2 FROM DENDROASPIS ANGUSTICEPS VENOM

Abstract A toxin, designated Ta2, has been isolated from the venom of the green mamba, Dendroaspis angusticeps, by ion exchange chromatography on Amberlite CG-50 and gel filtration through Sephadex G-50. Toxin Ta2 contains 60 amino acid residues, is devoid of phenylalanine and tryptophan, and is cross-linked by four intramolecular disulfide bridges. In its primary structure toxin Ta2 reveals homology with both the neuro- and cardiotoxins. Toxin Ta2 differs immunochemically completely from the long and short neurotoxins, cardiotoxins, and also from D. angusticeps toxin Ta1 (toxin FVII).

Snake Venom Toxins: The Evolution of Some of the Toxins Found in Snake Venoms

Strydom, D. J. (National Chemical Research Laboratory, Council for Scientific and Industrial Research, P. O. Box 395, Pretoria, South Africa) 1974. Snake Venom Toxins: The Evolution of some of the Toxins found in Snake Venoms. Syst. Zool. 22:596–608.—A computer program, ALIGN, was developed to test for homology of proteins. This program was used in conjunction with programs previously developed, to set up phylogenetic relationships and ancestral amino acid sequences for 43 snake venom toxins. From these and other studies emerged two mutually exclusive postulates for the evolution of snake venom toxins from digestive tract enzymes. Internal repitition in the sequence of toxins and homology of toxins with various enzymes were found. Evidence seems to favour one of the above postulates to a slight extent above the other. According to this it seems as if the short neurotoxins evolved from digestive tract enzymes like, for example, ribonuclease and that the cytotoxins and long neurotoxins evolved from the short neurotoxins. Other toxins have developed from, for example, trypsin inhibitors. The concepts of Darwinian and non-Darwinian evolution are both used to explain the evolution of the protein-toxins in snake venoms.