Isolation and primary structure of the major toxin from sea snake, Acalyptophis peronii, venom

The major neurotoxin from the venom of Acalyptophis peronii captured in the Gulf of Thailand was isolated. Although there are two toxic fractions in the venom, the most toxic and abundant fraction was selected for purification and chemical characterization. The LD50 of the major toxin is 0.125 micrograms/g mice, indicating an extremely toxic nature. The toxin consists of 60 amino acid residues with methionine as the amino-terminal and asparagine as the carboxy-terminal end. It contains nine half-cystine residues. There is 1 mol each of tryptophan, tyrosine, methionine, valine, aspartic acid, leucine, and alanine, and there is no phenylalanine. The molecular weight calculated from the amino acid sequence determination was 6600. The toxin replaces alpha-bungarotoxin in binding with the acetylcholine receptor, indicating that the A. peronii major neurotoxin competes with alpha-bungarotoxin for the same binding site of the acetylcholine receptor.     

Amino acid sequences of two novel long-chain neurotoxins from the venom of the sea snake Laticauda colubrina.

From the venom of a population of the sea snake Laticauda colubrina from the Solomon Islands, a neurotoxic component, Laticauda colubrina a (toxin Lc a), was isolated in 16.6% (A280) yield. Similarly, from the venom of a population of L. colubrina from the Philippines, a neurotoxic component, Laticauda colubrina b (toxin Lc b), was obtained in 10.0% (A280) yield. The LD50 values of these toxins were 0.12 microgram/g body wt. on intramuscular injection in mice. Toxins Lc a and Lc b were each composed of molecules containing 69 amino acid residues with eight half-cystine residues. The complete amino acid sequences of these two toxins were elucidated. Toxins Lc a and Lc b are different from each other at five positions of their sequences, namely at positions 31 (Phe/Ser), 32 (Leu/Ile), 33 (Lys/Arg), 50 (Pro/Arg) and 53 (Asp/His) (residues in parentheses give the residues in toxins Lc a and Lc b respectively). Toxins Lc a and Lc b have a novel structure in that they have only four disulphide bridges, although the whole amino acid sequences are homologous to those of other known long-chain neurotoxins. It is remarkable that toxins Lc a and Lc b are not coexistent at the detection error of 6% of the other toxin. Populations of Laticauda colubrina from the Solomon Islands and from the Philippines have either toxin Lc a or toxin Lc b and not both of them.     

Snake venomics of two poorly known Hydrophiinae: Comparative proteomics of the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis cyanocinctus

The venom proteomes of Toxicocalamus longissimus and Hydrophis cyanocinctus, a fossorial and a marine species, respectively, of the Hydrophiinae genus of Elapidae, were investigated by Edman degradation of RP-HPLC isolated proteins, and de novo MS/MS sequencing of in-gel derived tryptic peptide ions. The toxin arsenal of T. longissimus is made up of 1-2 type-I PLA(2) molecules, which account for 6.5% of the venom proteins, a minor PIII-SVMP (1.4% of the venom toxins), and ~20 members of the 3FTx family comprising 92% of the venom proteome. Seventeen proteins (5 type-I PLA(2)s and 12 3FTxs) were found in the venom of H. cyanocinctus. Three-finger toxins and type-I PLA(2) proteins comprise, respectively, 81% and 19% of its venom proteome. The simplicity of the H. cyanocinctus venom proteome is highlighted by the fact that only 6 venom components (3 short-chain neurotoxins, two long-chain neurotoxins, and one PLA(2) molecule) exhibit relative abundances >5%. As expected from its high neurotoxin abundance, the LD(50) for mice of H. cyanocinctus venom was fairly low, 0.132μg/g (intravenous) and 0.172μg/g (intraperitoneal). Our data indicate that specialization towards a lethal cocktail of 3FTx and type-I PLA(2) molecules may represent a widely adopted trophic solution throughout the evolution of Elapidae. Our results also points to a minimization of the molecular diversity of the toxin arsenal of the marine snake Hydrophis cyanocinctus in comparison to the venom proteome of its terrestrial relatives, and highlight that the same evolutionary solution, economy of the toxin arsenal, has been convergently adopted by different taxa in response to opposite selective pressures, loss and gain of neurotoxicity.     

Tarantula (Eurypelma californicum) venom, a multicomponent system

The venom of the tarantula Eurypelma californicum was analysed biochemically, the components were isolated and characterized. The pH value of the crude venom is 5.3 +/- 0.3. After dilution with distilled water, UV-absorption spectra showed a single maximum at 258 nm (pH ca. 7.0). A second maximum at 328 nm emerged above pH 8.0. Protein concentration of the venom is ca. 65 mg/ml. After Coomassie staining SDS-PAGE patterns show three major bands with apparent molecular masses around 40 kDa, 4.3 kDa and 1.3 kDa besides some weak high molecular protein bands. The following low-molecular mass constituents were determined in the crude venom: ATP, ADP, AMP, glutamic acid, aspartic acid, gamma-aminobutyric acid, glucose and the ions potassium, sodium, calcium, magnesium and chloride; the osmolality was 361 micro0smol/ml. The LD50 value for female cockroaches was 0.15 microliters venom per g body weight and for male cockroaches 0.4 microliters venom per g body weight. Separation of the crude venom by gel chromatography yielded four elution peaks. Peak I contains the enzyme hyaluronidase. The activity is 200-900 U/microliters. Peak II contains a mixture of toxic peptides. Peak III contains the 1.3-kDa components of SDS-PAGE and peak IV mainly contains ATP. Venom proteins including the enzyme hyaluronidase were precipitated by 5% trichloroacetic acid. The supernatant was separated by HPLC into 13 fractions. Fraction 1 contains glutamic acid, aspartic acid, gamma-aminobutyric acid and ATP; fraction 2 contains ATP, ADP and AMP as well as a component 2' visible in SDS-PAGE as 1.3-kDa band and consisting of spermine and tryptophan; fraction 3 contains ATP and an unknown component 3'; fractions 4-6 also show a 1.3-kDa band in SDS-PAGE, fraction 4 being tyrosylspermine and fractions 5 and 6 containing compounds of spermine and aromatic molecules; fraction 7 contains a peptide which lacks aromatic amino acids, it was sequenced from the N-terminus; fractions 8-13 contain very similar toxic peptides. The peptides in fractions 11 and 12, labeled ESTX for Eurypelma spider toxin, were cleaved with different enzymes and sequenced. They differ in one amino acid in position 26. Homologies with scorpion toxins and with a toxin of the spider Segestria florentina were found.     

Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis.

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 mug/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.     

Hornetin: the lethal protein of the hornet (Vespa flavitarsus) venom

By gel permeation on a Fractogel TSK HW 50 column followed by ion-exchange chromatography on carboxymethylcellulose CM 52, a lethal protein, designated hornetin, was purified from the venom of Vespa flavitarsus. Hornetin is a highly basic protein (pI 10.2) with a molecular mass of about 32 kDa. Its amino acid composition is characterized by a high content of lysine, aspartic and glutamic acid, and is devoid of tryptophan and cysteine. The lack of cysteine in the molecule is distinct from other known vespid venom proteins of comparable size. The i.v. LD50 of the toxin is 0.42 microgram per g mouse. Assayed on the red blood cells of the mouse and guinea-pig as well as isolated nerve muscle preparations of the chick and mouse, hornetin showed direct hemolytic activity and presynaptic neurotoxicity at microgram level and displayed musculotropic effect at higher concentrations.     

Plant pathotoxins from Alternaria citri: The minor ACRL toxins

Five host-specific pathotoxins, ACRL toxins II, III, III′, IV and IV′, were isolated from the culture broth of Alternaria citri, the fungus causing brown spot disease of rough lemon. These toxins are related structurally to the major ACRL toxin, toxin I, and to its derivative compound A. Chemical and spectral studies indicated that the ACRL minor toxins were a group of analogous compounds of different chain lengths all of which have a α-pyrone group, in contrast to the dihydro-α-pyrone group in toxin I. Toxin II showed a very low biological activity (ED₅₀ greater than 10 μg/ml) whereas the other minor toxins had slightly higher activities ranging from 1 to 10 μg/ml. The dihydropyrone group in ACRL toxin I was correlated with high biological activity (ED₅₀ = 18–30 ng/ml).     

A mammal toxin derived from the venom of a chactoid scorpion

1. It has been shown that the low toxicity to mammals (LD50 of about 200 mg per kg mice body weight) of the chactoid scorpion venom Scorpio maurus palmatus (Scorpionidae) is due to a single low molecular weight basic protein. 2. This compound was purified by the aid of gel filtration and ion exchange column chromatography, possessed about 80% of the mice lethality of the crude venom with an increase of about 60 fold in its specific toxicity. 3. It is composed of 32 amino acids (mol. wt = 3478) and devoid of isoleucine, leucine, phenylalanine, histidine and tryptophan. 4. The unique amino acid composition of the present toxin is compared to those of the well known buthoid scorpion venom mammal toxins and some other toxins derived from the same venom. 5. It is the first chemically characterized chactoid toxin.     

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.     

Pathological alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom

The pathological alterations induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi, were studied in mice. Neuwiedase was devoid of hemorrhagic activity when tested in the skin up to a dose of 200 microgram, and also after intramuscular injection in the gastrocnemius. However, it induced bleeding when applied onto the mouse cremaster muscle in intravital microscopy experiments, and caused pulmonary hemorrhage when injected intravenously at doses higher than 5 microgram/g. Median lethal dose (LD(50)) by the intravenous route was 5 microgram/g, whereas LD(50) of crude venom was 0.47 microgram/g. After intramuscular injection, neuwiedase induced a mild myotoxic effect, evidenced histologically and by the increment in plasma creatine kinase activity, but it was devoid of hemorrhagic and thrombotic effects. In contrast, crude B. neuwiedi venom induced prominent hemorrhage and myonecrosis in gastrocnemius muscle. Both venom and neuwiedase induced an inflammatory reaction in muscle tissue characterized by abundant polymorphonuclear leukocytes. Moreover, a conspicuous edema developed in the foot pad after subcutaneous injection of neuwiedase. Anti-neuwiedase antibodies produced in rabbits were effective in the neutralization of hemorrhagic activity of crude venom, evidencing immunological cross-reactivity between neuwiedase and other hemorrhagic metalloproteinases present in the venom, and suggesting that metalloproteinases devoid of, or having low, hemorrhagic activity could be good immunogens to generate antibodies effective against high molecular mass metalloproteinasas having potent hemorrhagic activity. It is concluded that neuwiedase, despite its lack of hemorrhagic effect when injected in the gastrocnemius muscle, contributes to local tissue damage by inducing edema, inflammatory infiltrate and mild myotoxicity, and by degrading extracellular matrix components. In addition, large doses of neuwiedase may contribute to pulmonary bleeding     

Isolation and primary structure of a potent toxin from the venom of the scorpion Centruroides sculpturatus Ewing.

A potent toxin has been purified from the venom of the scorpion Centruroides sculpturatus Ewing using the ion-exchange resin CM-Sepharose CL-6B at basic pH. The toxin, designated CsE M1, comprised 65 amino acid residues and its primary structure was established as: Lys-Glu-Gly-Tyr-Leu-Val-Asn-Ser-Tyr-Thr10-Gly-Cys-Lys-Tyr-Glu-Cys- Leu-Lys-Leu- Gly20-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Arg30-Gln-Gln-Tyr- Gly-Lys-Ser-Gly-Gly - Tyr-Cys40-Tyr-Ala-Phe-Ala-Cys-Trp-Cys-Thr-His-Leu50-Tyr-Glu- Gln-Ala-Val-Val-Trp - Pro-Leu-Pro60-Asn-Lys-Thr-Cys-Asn. CsE M1 is the most lethal protein to be identified in C. sculpturatus venom and the LD50 of the toxin, determined by subcutaneous injection into Swiss mice, is 87 micrograms/kg. CsE M1 shows strong structural similarity (92% positional identity) to the most potent beta-toxin, Css II, from the Mexican scorpion, Centruroides suffusus suffusus but is quite dissimilar to the previously characterized toxins with low potency isolated from C. sculpturatus Ewing.     

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.     

Snake venom toxins--I. The primary structure of a long neurotoxin S4C6 from Aspidelaps scutatus (shield or shield-nose snake) venom

1. Long neurotoxin S4C6 from Aspidelaps scutatus venom was purified by gel filtration and ion exchange chromatography (Joubert, 1987). 2. It contains 68 amino acids including 10 half-cystines. The toxicity of toxin S4C6 was determined and a LD50 of 0.13 +/- 0.04 micrograms/g mouse was found. 3. The complete primary structure of long neurotoxin S4C6 has been elucidated. In the toxin the 10 structurally invariant amino acids of the neurotoxins and cytoxins and the five functionally invariant amino acids of the neurotoxins are conserved.     

Purification and characterization of the cytotoxic Cerebratulus A toxins

Mucus secreted from the skin of a marine worm, Cerebratulus lacteus, contains a family of polypeptide cytotoxins (A toxins) in addition to the previously reported polypeptide neurotoxins (B toxins). The A toxins were purified by Sephadex G-50 chromatography and then CM-cellulose gradient chromatography at pH 7.5 and pH 3.5. The three most abundant A toxins (designated according to their order of CM-cellulose elution) were homogeneous by gel electrophoreses, amino acid composition, and by NH2-terminal and COOH-terminal partial sequence analyses. Each of the three A toxins consists of a single basic polypeptide chain of 93 to 99 residues, cross-linked by three or four disulfide bonds, lacking reducing sugar and cysteinyl residues. The three A toxins rapidly lysed human red cells and Ehrlich ascites tumor cells at 1 to 10 microgram/ml concentrations. On a molar basis toxin A-III is about 4 times more active than melittin (bee venom lysin) and over 10 times more active than cardiotoxin (elapid snake lysin) upon human red cells. Purified A toxins lacked phospholipase A activity. The cytoxins as well as the neurotoxins were concentrated within the body wall integument.     

Purification and partial characterization of stonustoxin (lethal factor) from Synanceja horrida venom.

1. The lethal factor of the stonefish (Synanceja horrida) venom, designated as the stonustoxin, was purified to homogeneity by a two-step procedure on Sephacryl S-200 High Resolution (HR) gel permeation and DEAE Bio-Gel A anion exchange chromatography. 2. Stonustoxin has a native mol. wt of 148,000 and an isoelectric point of 6.9. 3. SDS-polyacrylamide gel electrophoresis revealed two subunits (designated alpha and beta) with mol. wts of 71,000 and 79,000, respectively. 4. The amino acid composition of both subunits and the N-terminal amino acid sequence of the beta subunit were also determined. 5. Purified stonustoxin had an LD50 of 0.017 microgram/g which is 22-fold more potent than that of the crude venom. 6. The toxin exhibited potent haemolytic activity in vitro and edema-inducing activity with a minimum edema dose (MED) of 0.15 micrograms in mouse paw. The edema effect was not antagonized by diphenhydramine.     

Isolation, properties and amino acid sequence of a long-chain neurotoxin, Acanthophis antarcticus b, from the venom of an Australian snake (the common death adder, Acanthophis antarcticus).

The venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus), was chromatographed on a CM-cellulose CM52 column. One of the neurotoxic components, Acanthophis antarcticus b (toxin Aa b) was isolated in about 9.4% (A280) yield. The complete amino acid sequence of toxin Aa b was elucidated. Toxin Aa b is composed of 73 amino acid residues, with ten half-cystine residues, and has a formula weight of 8135. Toxin Aa b has no histidine or methionine residue in its sequence. The amino acid sequence of toxin Aa b is homologous with those of other neurotoxins with known sequences, although it is novel in having a valine residue at its N-terminus and an arginine residue at position-23, where a lysine residue is found in almost all the so-far-known neurotoxins. Irrespective of the latter replacement, the toxin Aa b is fully active, with an LD50 value (in mice) of 0.13 microgram/g body weight on intramuscular injection.     

In vivo and in vitro protection against lethal activity of Buthus occitanus tunetanus venom with a recombinant protein

We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Identification,cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Purification and characterization of insecticidal toxins from venom glands of the parasitic wasp, Bracon hebetor

The potency of venom from Bracon hebetor against lepidopterous larvae has been known for over 40 years, but previous attempts to purify and characterize individual protein toxins have been largely unsuccessful. Three protein toxins were purified from venom of this small parasitic wasp and the amino acid sequences of 22–31 consecutive residues at the amino-terminus were determined. These relatively large toxins (apparent molecular mass 73 kDa) were labile under many isolation techniques, but anion-exchange chromatography allowed purification with retention of biological activity. Two purified toxins were quite insecticidal (LD₅₀ < 0.3μg/g) when injected into six species of lepidopterous larvae. On a molar basis, one toxin (Brh-I) has the highest known biocidal activity against Heliothis virescens (LD₅₀ = 2 pmol/g).