Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.     

Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The water-soluble part of the dried venom from the scorpion, Tityus serrulatus Lutz and Mello (range, Southeastern Brazil), showed 16 polypeptide bands on polyacrylamide gel electrophoresis. This material exhibited toxic and hyaluronidase activity but no phospholipase, phosphodiesterase, protease, or fibrinolytic activity. Fractionation on glycinamide-treated Sephadex G-50 afforded three protein fractions, which were non-toxic, equitoxic, and three times more toxic than the water-soluble venom. Subsequent separation of the toxic fractions on carboxymethyl-cellulose with phosphate buffers furnished five toxic components, which were further purified on carboxymethyl-cellulose with a salt gradient in acetate buffer. Toxin γ, the major and most basic toxin, is a 62-residue protein that, unlike other scorpion toxins, contains methionine. Automated Edman degradation showed the amino-terminal sequence to be H-Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-. Toxin γ is the first example of a fifth structural type of mammalian toxin from scorpion venom. Its amino-terminal sequence shows greater homology with toxins similar to Centruroides suffusus suffusus toxin III and Androctonus australis toxin II than with toxins similar to A. australis toxin I or Bhutus occitanus tunetanus toxin I.     

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian 'armed' spider Phoneutria nigriventer

A lethal neurotoxic polypeptide of Mr 8 kDa was purified from the venom of the South American 'armed' or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S-carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.     

Isolation and amino acid sequence of a neurotoxic phospholipase A from the venom of the Australian tiger snake Notechis scutatus scutatus.

The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.     

The covalent structure of calf skin type III collagen. V. The amino acid sequence of the cyanogen bromide peptide alpha 1(III)CB9A (position 789 to 927).

The cyanogen bromide peptide alpha 1(III)CB9A is 139 amino acid residues in length and occupies positions 789--927 along the alpha 1(III) chain. Peptides necessary for the complete sequence analysis were obtained after fragmentation of alpha 1(III)CB9B with trypsin, protease from Staphylococcus aureus V8, hydroxylamine and chymotrypsin. They were separated mainly by chromatography on Sephadex G-50 and phosphocellulose and subsequently sequenced using the automated Edman degradation procedure.     

Phaiodotoxin, a novel structural class of insect-toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus.

A peptide called phaiodotoxin was isolated from the venom of the scorpion Anuroctonus phaiodactylus. It is lethal to crickets, but non toxic to mice at the doses assayed. It has 72 amino acid residues, with a molecular mass of 7971 atomic mass units. Its covalent structure was determined by Edman degradation and mass spectrometry; it contains four disulfide-bridges, of which one of the pairs is formed between cysteine-7 and cysteine-8 (positions Cys63-Cys71). The other three pairs are formed between Cys13-Cys38, Cys23-Cys50 and Cys27-Cys52. Comparative sequence analysis shows that phaiodotoxin belongs to the long-chain subfamily of scorpion peptides. Several genes coding for this peptide and similar ones were cloned by PCR, using cDNA prepared from the RNA of venomous glands of this scorpion. Electrophysiological assays conducted with this toxin in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ currents. However, it shifts the voltage dependence of activation and inactivation of insect Na+ channels (para/tipE) to more negative and positive potentials, respectively. Therefore, the 'window' current is increased by 225%, which is thought to be the cause of its toxicity toward insects. Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae.     

Proteomics of the venom from the Amazonian scorpion Tityus cambridgei and the role of prolines on mass spectrometry analysis of toxins

Scorpion venom are complex mixtures of peptides, known to cause impairment of ion-channel function in biological membranes. This report describes the separation of approximately 60 different components by high performance liquid chromatography and the characterization by Edman degradation and mass spectrometry of 26 peptides from the soluble venom of the Amazonian scorpion Tityus cambridgei. One of these peptides, named Tc48a, was fully characterized. It contains 65 amino acid residues, the C-terminal residue is amidated and it affects Na(+)-channels with a K(d) of about 82 nM. Furthermore, this report shows the thermo-instability of scorpion toxins subjected to electron spray ionization-mass spectrometry (ESI-MS). When a proline residue is located near the N-terminal region of the toxin, not stabilized by disulfide bridges, artificial components are generated by the mass spectrometer conditions, due to the cleavage of the peptide bond at the proline positions. This phenomenon was confirmed by using four model proteins (variable regions of immunoglobulins) studied by ESI-MS and matrix assisted laser desorption ionization-time of flight (MALDI-TOF)/MS.     

Amino-acid sequence of toxin III of Naja haje.

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja nivea alpha (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).     

Purification, characterization and molecular modelling of two toxin-like proteins from the Androctonus australis Hector venom.

Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins. In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion alpha-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.     

Hainantoxin-Ⅱ的分离纯化及其结构与功能分析(英文)

从分布于我国海南省的海南捕鸟蛛(Haplopelma hainanum)毒素中,利用阳离子交换色谱和反相高效液相色谱分离得到一种多肽神经毒素,命名为Hainantoxin-Ⅱ(HnTx-Ⅱ)。MALDI-TOF质谱分析表明该多肽毒素相对分子质量为4253.135,其氨基酸序列经Edman降解测序为LFECS VSCEI EKEGN KDCKK KKCKG GWKCK FNMCV KV,其中的6个Cys形成3对二硫键。同源性搜索表明,HnTx-Ⅱ与Huwentoxin-Ⅱ(HwTx-Ⅱ)的同源性高达91%,仅有3个氨基酸残基不同。HwTx-Ⅱ为从虎纹捕鸟蛛中提取的杀虫肽,该多肽具有独特的结构模体。活性分析表明HnTx-Ⅱ与HwTx-Ⅱ具有相似的生物学活性。经腹腔注射HnTx-Ⅱ,美洲蜚蠊可被麻痹,其ED50为16μg/g;而当剂量增加到60μg/g时,可立即杀死美洲蜚蠊,其杀死昆虫活性明显强于HwTx-Ⅱ。此外,HnTx-Ⅱ经脑室注射可杀死小鼠,其LD50为1.41μg/g,该活性却明显低于HwTx-Ⅱ。这两种多肽毒素的结构与功能的差异为进一步阐明多肽毒素的结构与功能之间的关系提供良好的研究模型。     

Two-dimensional 1H nuclear magnetic resonance study of AaH IT, an anti-insect toxin from the scorpion Androctonus australis Hector. Sequential resonance assignments and folding of the polypeptide chain

Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includes two and a half turns of alpha-helix running from residues 21 to 30 and a three-stranded antiparallel beta-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the alpha-helix to an external strand of the beta-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. These structure elements are very similar to the secondary structure reported in single crystals for either variant 3 from the scorpion Centruroides sculpturatus Ewing (CsE V3) or toxin II from the scorpion A. australis Hector (AaH II). The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. This surface is made of an aromatic cluster that is surrounded by long hydrophobic side-chain residues, as well as the loops protruding out of it. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction.     

Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies.

A synthetic gene encoding the Group II phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA2's were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA2. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA2 demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.     

Discrepin, a new peptide of the sub-family alpha-ktx15, isolated from the scorpion Tityus discrepans irreversibly blocks K+ -channels (IA currents) of cerebellum granular cells

A new peptide was purified from the venom of the Venezuelan scorpion Tityus discrepans, by high-performance liquid chromatography and its amino acid sequence was completed by Edman degradation and mass spectrometry analysis. It contains 38 amino acid residues with a molecular weight of 4177.7 atomic mass units, tightly folded by three disulfide bridges, and has a pyroglutamic acid at the N-terminal region. This peptide, named Discrepin, was shown to block preferentially the IA currents of the voltage-dependent K+ -channel of rat cerebellum granular cells in culture. The K+ -currents are inhibited in an apparently irreversible manner, whose 50% inhibitory effect is reached with a 190 nM toxin concentration. The systematic nomenclature proposed for this toxin is alpha-KTx15.6.     

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.     

A novel short neurotoxin,cobrotoxin c,from monocellate cobra (Naja kaouthia) venom: isolation and purification,primary and secondary structure determination,and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).     

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.     

Cloning, sequence analysis and homology modeling of a novel phospholipase A2 from Heterometrus fulvipes (Indian black scorpion).

We report the cloning and sequencing of group III phospholipaseA(2) from Heterometrus fulvipes (HfPLA(2)), Indian black scorpion. The cDNA sequence codes for the mature portion of the group PLA(2) of 103 amino acids. The sequence has 85% identity with Mesobuthus tamulus (Indian red scorpion) PLA(2) and a 40% identity with bee venom PLA(2) and human group III PLA(2). Most of the essential features of group III PLA(2) like Ca(2+) binding loop and catalytic residues are conserved. Homology modeling was done with the known structure of group III bee venom PLA(2). All the secondary structural motifs and the disulfide bridges are as predicted. The variation like the replacement of aspartic acid residue with glutamic acid in the well known histidine-aspartic acid dyad is a rare feature. This is the first structural model report of an Indian black scorpion PLA(2).     

Isolation and characterization of a hyaluronidase from the venom of Chinese red scorpion Buthus martensi

A hyaluronidase, named BmHYA1, was purified from the venom of Chinese red scorpion (Buthus martensi), using successive chromatography. The homogeneity of BmHYA1 was confirmed by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular mass of BmHYA1 was 48,696 Da determined by MALDI-TOF MS. The optimal temperature and pH of BmHYA1 were 50 degrees C and pH 4.5, respectively. It could be inhibited by DTT, Cu(2+), Fe(3+) or heparin, but not Mg(2+), Ca(2+), reduced glutathione, l-cysteine or EDTA. The sequence of thirty N-terminal amino acids of BmHYA1 was obtained by Edman degradation, as TSADF KVVWE VPSIM CSKKF KICVT DLLTS; but no similarity was found to other venom hyaluronidases. Further, BmHYA1 can hydrolyze hyaluronan into relatively smaller oligosaccharides and modulate the expression of CD44 variant in the breast cancer cell line MDA-MB-231.     

The beta-type toxin Ts II from the scorpion Tityus serrulatus: amino acid sequence determination and assessment of biological and antigenic properties.

The toxin Ts II from the venom of the Brazilian scorpion Tityus serrulatus was purified in two successive chromatographic steps. The amino acid sequence was then determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of proteolytic peptides derived from it. This sequence appears to differ from that of previously characterized toxins found in this venom. However, it is identical to the recently published sequence of protein III-8 from the same venom [Possani et al., J Biol Chem 266:3178-3185, 1991], except that the C-terminus was found to be amidated. Homologies were found between the sequence of Ts II and that of other toxins from Tityus; in particular, the amino acid sequence of Ts II displays 72% sequence identity with Ts VII (also called Titx gamma). Consistent with this structural similarity, some biological properties of Ts II were found to be similar to those of Ts VII: Ts II has an intracerebroventricular LD50 of 6 ng, as compared to 0.6 ng for Ts VII; in a receptor binding assay Ts II, like Ts VII, was found to behave as a beta-type toxin and to inhibit the binding of the reference labelled toxin with a K0.5 of 5 x 10(-9) M, as compared to 7 x 10(-11) M for Ts VII. Nevertheless, Ts II is unable to bind to anti-Ts VII antibodies in radioimmunoassay experiments, indicating the non-conservation between the two toxins of at least some antigenically important residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius

A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68 Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42 Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I-III, II-IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0 nmol) into mice, peptide sr5a caused depressed behavioral activity.