Studies on the subunit structure of textilotoxin, a potent neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis)

Textilotoxin is a presynaptic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis. It has the highest lethality and is structurally the most complex of any known snake venom neurotoxin. It was resolved into its five non-covalently linked subunits in a single step by reverse-phase HPLC. Two of the subunits were identical. The N-terminal amino-acid sequence and amino-acid composition of each subunit were determined. Subunit A was the only one found to possess phospholipase A activity. Separation of textilotoxin into its subunits was reversible and reformed textilotoxin had the same Mr and lethality in mice as the native toxin. Experiments with various unnatural combinations of subunits have led to interesting variations in lethality and Mr of the resulting complexes.     

Pseudonajatoxin b: unusual amino acid sequence of a lethal neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis

The complete amino acid sequence of pseudonajatoxin b, a basic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis, was determined by automated Edman analysis of the reduced carboxymethylated polypeptide and of peptides derived by digestion of it with Staphylococcus aureus V8 proteinase. Pseudonajatoxin b consists of a single polypeptide chain of 71 amino acids with Mr 7762. The amino acid sequence showed considerable homology with postsynaptic long neurotoxins, but there were striking differences. Pseudonajatoxin b displayed relatively high lethality, LD50 15 micrograms/kg in mice.     

The complete amino acid sequence of a post-synaptic neurotoxin isolated from the venom of the Australian death adder snake Acanthophis antarcticus

1. A lethal neurotoxin (acanthophin d) was isolated from the venom of the Australian death adder snake Acanthophis antarcticus. 2. Acanthophin d consisted of a single polypeptide chain of 74 amino acid residues cross-linked by five disulphide bridges. 3. The results of neurophysiological experiments on murine phrenic nerve hemi-diaphragm preparations were consistent with irreversible post-synaptic blockage of neuromuscular transmission by acanthophin d.     

Amino-acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus)

The amino acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan, has been determined. The beta 1 isosubunit, which is neither toxic nor enzymatically active on its own, consists of a single polypeptide chain of 118 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin beta 1 isosubunit is closely homologous to the taipoxin alpha and gamma subunits and to enzymatically active pancreatic and elapid snake venom phospholipases A2.     

Amino acid sequence of kappa-flavitoxin: establishment of a new family of snake venom neurotoxins

The complete amino acid sequence of kappa-flavitoxin, a neurotoxin isolated from the venom of Bungarus flaviceps, has been determined by automated Edman analysis of the intact protein and of peptides derived from digests with trypsin and chymotrypsin. kappa-Flavitoxin consists of a single 66-residue polypeptide chain which is completely devoid of methionine. The amino acid sequence of kappa-flavitoxin demonstrates that although the toxin is related to the alpha-neurotoxin family, it displays a much higher degree of homology with kappa-bungarotoxin. The conserved structural features of the kappa-neurotoxins and their pharmacological profiles, which are distinct from those of all known alpha-neurotoxins, provide evidence for a new, structurally and functionally unique family of snake venom neurotoxins.     

A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization

A novel prothrombin activator, Mikarin, has been isolated from Micropechis ikaheka venom. It is a single polypeptide chain metalloproteinase with the apparent molecular weight of 47kDa. Mikarin exhibits Ca(2+)-independent prothrombin activation, but no effects on other blood coagulation factors, such as factor X and fibrinogen. Mikarin is the first member of group I prothrombin activators from elapid venom. Like other high-molecular-weight snake venom proteinases, it has three structural domains, metalloproteinase and disintegrin-like and Cys-rich domains, and belongs to the P-III class of snake venom metalloproteinases. The N-terminal of Mikarin exhibits 76% sequence identity with Cobrin, a metalloproteinase identified from Naja naja venom, but very lower identities were found when compared with those from viperid and crotalid venom. In addition, the presence of disintegrin-like and Cys-rich domains in snake venom metalloproteinases with diverse biological activities suggests that these domains may be important for their function.     

Identification by cross-linking of a beta-bungarotoxin binding polypeptide in chick brain membranes.

beta-Bungarotoxin (beta-BTX) is a snake venom neurotoxin which inhibits neurotransmitter release from different types of nerve terminals. To identify presynaptic membrane components potentially important in neurosecretion, 125I-labeled beta-BTX (mol. wt. 21 000) was cross-linked to a high-affinity binding site in synaptic membrane fractions of chick brain using the photoactivable cross-linker N-succinimidyl-6(4'-azido-2'-nitrophenylamino)-hexanoate. Electrophoretic analysis of the cross-linked membrane proteins under both reducing and non-reducing conditions revealed a single [125I]beta-BTX-polypeptide adduct of apparent mol. wt. 116 000 (+/- 2000). The labeling of this band was prevented under conditions previously shown to inhibit the binding of [125I]beta-BTX to its high-affinity binding site. It is concluded that the cross-linking procedure identified a polypeptide of the presynaptic binding site for beta-BTX, and that this polypeptide has a mol. wt. of 95 000.     

Isolation, toxicity and amino terminal sequences of three major neurotoxins in the venom of Malayan krait (Bungarus candidus) from Thailand

We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.     

Isolation and characterization of a novel postsynaptic/cytotoxic neurotoxin from Daboia russelli russelli venom.

A postsynaptic neurotoxin was purified from Daboia russelli russelli venom using gel filtration, ion-exchange chromatography and reverse-phase high-performance liquid chromatography. The N-terminal sequence, molecular mass and pharmacological activities of the neurotoxin/cytotoxin indicate that it is a short-chain neurotoxin like that found in Elapid venom. This is the first report on the presence of such a postsynaptic neurotoxin from D. r. russelli venom.     

Comparison of three classes of snake neurotoxins by homology modeling and computer simulation graphics

We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.     

Sequence analysis and phylogenetic study of some toxin proteins of snakes and related non-toxin proteins of chordates

Snakes are equipped with their venomic armory to tackle different prey and predators in adverse natural world. The venomic composition of snakes is a mix of biologically active proteins and polypeptides. Among different components snake venom cytotoxins and short neurotoxin are non-enzymatic polypeptide candidates with in the venom. These two components structurally resembled to three-finger protein superfamily specific scaffold. Different non-toxin family members of three-finger protein superfamily are involved in different biological roles. In the present study we analyzed the snake venom cytotoxins, short neurotoxins and related non-toxin proteins of different chordates in terms of amino acid sequence level diversification profile, polarity profile of amino acid sequences, conserved pattern of amino acids and phylogenetic relationship of these toxin and nontoxin protein sequences. Sequence alignment analysis demonstrates the polarity specific molecular enrichment strategy for better system adaptivity. Occurrence of amino acid substitution is high in number in toxin sequences. In non-toxin body proteins there are less amino acid substitutions. With the help of conserved residues these proteins maintain the three-finger protein scaffold. Due to system specific adaptation toxin and non-toxin proteins exhibit a varied type of amino acid residue distribution in sequence stretch. Understanding of Natural invention scheme (recruitment of venom proteins from normal body proteins) may help us to develop futuristic engineered bio-molecules with remedial properties.     

Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.     

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 Ca²⁺ 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.     

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.     

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.     

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of the inhibitor of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes)

We are presenting the first primary structure of a snake venom inhibitor. It was isolated from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) which represents a complex of a strong toxic basic protein with phospholipase A2 activity (2 isoenzymes) and the nontoxic acidic component functioning as its inhibitor. The sequence was established by automatic degradation in a liquid phase sequenator on the S-carboxymethylated chain and on the peptides obtained by tryptic hydrolysis of the oxidized chain. A limited tryptic digestion of the oxidized chain provided the necessary overlapping peptides. The inhibitor consists of 122 amino-acid residues including 14 cysteine and 10 tyrosine residues and is thus similar to the phospholipases from snake venoms. A comparison of the inhibitor sequence with the primary structure of the phospholipase A2 (CM-II) from the Horned Adder (Bitis nasicornis) venom shows a surprising homology of 52%. The identical amino acids include the cysteine and tyrosine residues and are generally accumulated in the surroundings of cysteine residues. The histidine (pos. 47) in the active center of the phospholipase A2 is substituted by glutamine in the inhibitor, but the tryptophan (pos. 30) which is essential for the enzymatic activity is present. The significant homology between enzyme and inhibitor in the vipoxin complex is believed to originate from a gene duplication. The relatively late development of the reptiles and the snake venom complex explains the highly preserved structure compared to other enzyme-inhibitor systems.     

Antisnake venom activity of ethanolic seed extract of Strychnos nux vomica Linn

The whole seed extract of S. nux vomica (in low doses) effectively neutralized Daboia russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist.     

Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates

Snake venom contains a diverse array of proteins and polypeptides. Cytotoxins and short neurotoxins are non-enzymatic polypeptide components of snake venom. The three-dimensional structure of cytotoxin and short neurotoxin resembles a three finger appearance of three-finger protein super family. Different family members of three-finger protein super family are employed in diverse biological functions. In this work we analyzed the cytotoxin, short neurotoxin and related non-toxin proteins of other chordates in terms of functional analysis, amino acid compositional (%) profile, number of amino acids, molecular weight, theoretical isoelectric point (pI), number of positively charged and negatively charged amino acid residues, instability index and grand average of hydropathy with the help of different bioinformatical tools. Among all interesting results, profile of amino acid composition (%) depicts that all sequences contain a conserved cysteine amount but differential amount of different amino acid residues which have a family specific pattern. Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins. Different biological system dependent adaptation gives the birth of enriched bio-molecules. Understanding of physicochemical properties of these proteins will help to generate medicinally important therapeutic molecules for betterment of human lives.     

重组α-银环蛇毒素的纯化及活性分析

以表达重组α-银环蛇毒素的高效表达菌株BL21(PDZ04)为材料,研究重组α-银环蛇毒素(α-bungarotoxin,α-BgTx)的分离纯化.采取亲和色谱和离子交换色谱的方法都得到了重组α-银环蛇毒素的纯品.首先从天然的银环蛇毒干粉中分离纯化得到了天然α-银环蛇毒素的纯品.然后以天然α-银环蛇毒素为对照来检测重组α-银环蛇毒素的抗原性和毒性.ELISA结果显示其具有与天然α-银环蛇毒素相似的抗原性,以小鼠为动物模型,纯化的重组α-银环蛇毒素与天然α-银环蛇毒素相比,其腹腔注射的LD50也基本一致,约为0.22μg/g.结果表明利用基因工程的方法生产蛇神经毒素是可行的.