Complete amino acid sequences of two protease inhibitors in the venom of Bungarus fasciatus

Two analogous protease inhibitors, VIIIb and IX in the venom of Bungarus fasciatus were reduced and carboxymethylated. Tryptic peptides were separated by cellulose thin-layer peptide mapping technique, and amino acid sequences were analyzed by DABITC/PITC double coupling method. Alignment of all tryptic peptides was established by analyses of chymotryptic peptides and further confirmed by Staphylococcus aureus V8 protease digestion. IX consisted of 65 amino acid residues. VIIIb consisted of 62 residues, identical to the N-terminal 62-amino acid sequence of IX.     

Phospholipase A2 activity of long-chain cardiotoxins in the venom of the banded krait (Bungarus fasciatus)

Reinvestigation of the long-chain cardiotoxins from Bungarus fasciatus venom reveals that they are weak phospholipases of the A₂ type. The specific activities (units/mg) toward egg lecithin are 0.42, 1.65, and 0.29 for the long-chain cardiotoxins V-2, V-3 and VI, respectively.     

Complete amino acid sequence of a phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The acidic phospholipase A₂ has been isolated from Naja naja atra (Taiwan cobra) venom. The reduced and S-carboxymethylated enzyme was digested with trypsin, and eleven tryptic peptides which accounted for the whole molecule were isolated by a combination of chromatographic and gel filtration procedures, and their amino acid sequences were determined. The alignment of those eleven tryptic peptides was established by the analysis of nine major peptides obtained by Staphylococcus aureus protease digestion of the reduced and S-carboxymethylated enzyme. The phospholipase A₂ of Naja naja atra is a single polypeptide chain consisting of 120 amino acid residues including 14 intramolecularly linked half-cystines. It shows about 86% (103 out of 120) homology when compared with the Naja mossambica mossambica CM-II enzyme.     

Isolation and characterization of a presynaptic neurotoxin,P-elapitoxin-Bf1a from Malaysian Bungarus fasciatus venom

Presynaptic neurotoxins are one of the major components in Bungarus venom. Unlike other Bungarus species that have been studied, β-bungarotoxin has never been isolated from Bungarus fasciatus venom. It was hypothesized that the absence of β-bungarotoxin in this species was due to divergence during evolution prior to evolution of β-bungarotoxin. In this study, we have isolated a β-bungarotoxin isoform we named P-elapitoxin-Bf1a by using gel filtration, cation-exchange and reverse-phase chromatography from Malaysian B. fasciatus venom. The toxin consists of two heterogeneous subunits, subunit A and subunit B. LCMS/MS data showed that subunit A was homologous to acidic phospholipase A₂ subunit A3 from Bungarus candidus and B. multicinctus venoms, whereas subunit B was homologous with subunit B1 from B. fasciatus venom that was previously detected by cDNA cloning. The toxin showed concentration- and time-dependent reduction of indirect-twitches without affecting contractile responses to ACh, CCh or KCl at the end of experiment in the chick biventer preparation. Toxin modification with 4-BPB inhibited the neurotoxic effect suggesting the importance of His-48. Tissue pre-incubation with monovalent B. fasciatus (BFAV) or neuro-polyvalent antivenom (NPV), at the recommended titer, was unable to inhibit the twitch reduction induced by the toxin. This study indicates that Malaysian B. fasciatus venom has a unique β-bungarotoxin isoform which was not neutralized by antivenoms. This suggests that there might be other presynaptic neurotoxins present in the venom and there is a variation in the enzymatic neurotoxin composition in venoms from different localities.     

Amino acid sequence of a neutral phospholipase A2 (III) in the venom of Bungarus fasciatus

Two phospholipases were found in the venom of Bungarus fasciatus, one in fraction III, the other in fraction X of the chromatographic separation. A neutral PLA₂(III) purified from fraction III was subjected to amino acid sequencing by means of an automated sequenator applied to the intact RCM-PLA₂ (III) and the individual peptides obtained from HPLC separation of the three types of enzymatic peptides. PLA(III) was shown to consist of 118 amino acid residues with 14 half-cystines. It is 65% homologous to the basic PLA₂ obtained from fraction X.     

Covalent structure of toxins I and II from the scorpion Buthus occitanus tunetanus

The amino acid sequences of neurotoxins I and II, which are active on mammals, purified from the venom of Buthus occitanus tunetanus have been determined using standard methods, including mainly automatic phenylisothiocyanate degradation of S-carboxymethylated derivatives of the two proteins and peptides derived by enzymatic hydrolyses. Both toxins are made of sixty-five amino acid residues cross-linked with four disulfide bridges. For toxin II, the complete covalent structure, including the positions of the four disulfide bridges was determined: the positions are similar to those previously found in toxin II of another scorpion from Africa, Androctonus australis Hector. This finding is in favor of a similar structure for all of the scorpion neurotoxins active on mammals.     

A study of cardiotoxic principles from the venom of Bungarus fasciatus (Schneider).

Two cardiotoxin-like fractions (VI A and VI B) were isolated from the venom of Bungarus fasciatus (Schneider) by means of column chromatography on CM cellulose. The homogeneity of these two fractions was verified by microzone and disc electrophoresis. These two fractions shared with cobra cardiotoxin (CTX) many pharmacological actions. Both toxins produced skeletal muscle contracture, depression on cardiac muscle, blockade of neuromuscular transmission, local irritation and had anticholinesterase activity. However, the molecular weights of VI A and VI B were about two times that of CTX and the amino acid composition was also quite different from that of CTX. Moreover, both VI A and VI B were devoid of direct hemolytic action.The contracture inducing activity of VI B and CTX could be abolished by EDTA or high Ca²⁺ (12 mM) and accelerated by low Ca²⁺ (0.5 mM) medium. However, a high Mg²⁺ (10 mM) medium inhibited VI B induced contracture but accelerated CTX contracture. Spermine, spermidine and protamine were without effect on VI B contracture but accelerated profoundly CTX induced contracture. From these results, it is considered that the mode of contracture inducing activity of VI B may be different from that of CTX.     

Isolation of “ceruleotoxin” from Bungarus fasciatus venoms

Ceruleotoxin is a potent neurotoxin which irreversibly blocks the neuromuscular transmission at a postsynaptic level, without preventing the binding of acetylcholine to its receptor. We have originally purified this toxin from a batch of venom obtained from the Pasteur Institute, which was thought to be Bungarus caeruleus venom. Recently Noble and co-workers have observed that the protein composition of Bungarus caeruleus provided by Miami Serpentarium significantly differed from that of the Pasteur Institute batch, which they concluded therefore to be of a different origin.In order to clarify this point, the venom composition of various Bungarus species from several origins have been analysed by electrophoresis and by electrofocusing on polyacrylamide gels. Although individual variations exist between samples of the same snake species, the venom from Bungarus caeruleus, Bungarus fasciatus and Bungarus multicinctus possess distinct and characteristic protein compositions. The results of this study allowed us to identify unambiguously the batch used to purify the ceruleotoxin, as a Bungarus fasciatus venom.We identified a neurotoxin similar to ceruleotoxin in each of the five samples of Bungarus fasciatus venoms that we tested. On the contrary we did not detect such a toxin either in Bungarus caeruleus or in Bungarus multicinctus venoms. All purified ceruleotoxins are acidic proteins with a high toxicity (their LD₅₀ by intravenous injection in mice are from 0.04 to 0.06 mg per kg), which irreversibly block the postsynaptic response of Electrophorus electricus electroplaque to cholinergic agonists. They also possess a phospholipase A₂ activity (200 nmoles of egg lecithins hydrolysed per min per mg of protein). In this respect, ceruleotoxin is analogous to crotoxin and β-bungarotoxin.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

Amino acid sequences of phospholipases A2 from the venom of an Australian elapid snake (king brown snake, Pseudechis australis)

Two basic phospholipases A2 (Pa-11 and Pa-13) have been isolated from the venom of an Australian elapid snake, Pseudechis australis (king brown snake). The reduced and S-carboxymethylated phospholipases A2 were digested with trypsin and the resulting peptides were purified by a combination of chromatography on a DEAE-cellulose DE-52 column and gel filtration procedures. Eleven main peptides from Pa-11 and 9 peptides from Pa-13 could account for the amino acid compositions of the respective enzyme molecules. The alignment of the tryptic peptides and unelucidated regions of the amino acid sequences of tryptic peptides were established by the analysis of the peptides obtained by chymotryptic and/or Staphylococcal protease digestions. Each phospholipase A2 consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. Although Pa-11 is enzymatically 30-times as active as Pa-13 and highly toxic as compared to Pa-13, they are highly homologous in their amino acid sequences. They are also homologous to the enzymes from mammalian pancreas and the other snake venom phospholipases A2, especially to those from snakes belonging to the subfamilies Acanthophiinae and Laticaudinae.     

Molecular cloning of the major lethal toxins from two kraits (Bungarus flaviceps and Bungarus candidus).

The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.     

Nitration of tyrosine in three cobra neurotoxins

The curarumimetic snake venom toxins always have a tyrosine residue in the same position in their sequences. This invariant tyrosine in three neurotoxins has been nitrated with tetranitromethane. The toxins were: (1) siamensis 3 of the structural type 71-5 (no. of amino acid residues and disulfides) and with one tyrosine, the main neurotoxin of the venom of Naja naja siamensis, (2) siamensis 7C (62-4 and two tyrosines), a minor neurotoxic component of the same venom, and (3) toxin α (61-4 and one tyrosine), the main neurotoxin of Naja nigricollis. The first toxin is a so-called long neurotoxin (a toxin with five disulfides and ca. 70 amino acid residues) and the other two short toxins (four and ca. 60).The 3-nitrotyrosyl derivatives of siamensis 3 and toxin α were isolated by gel filtration on Sephadex G-50 and ion-exchange chromatography on Bio-Rex 70. Both derivatives had i.v. ld₁₀₀-doses of 150 μg per kg mouse, corresponding to 67% of the initial toxicity. This high residual activity indicates that the invariant tyrosine does not have an essential role neither as a stabilizer of the active conformation nor as a functional group. The nitration of toxin 7C involved the modification of both tyrosine residues and was accompanied by great structural changes and loss of activity. The inactivation does not imply that the invariant tyrosine should be functionally essential in this toxin, but it is rather a consequence of the serious conformational changes accompanying the modification.The pK_app of the invariant tyrosine is 10·5 in siamensis 3 and 11·6 in toxin α and probably about the same in toxin 7C. This indicates that this tyrosine in the long neurotoxin has an exposed position, whereas it is in the hydrophobic interior of the two short toxins, or that its ionization is greatly affected by neighbouring groups.In the discussion it is pointed out that the very strong binding between a neurotoxin and its target, the acetylcholine receptor is probably a result of the interaction between several groups in the toxin and the receptor. The majority of amino acid residues with reactive groups in the side chains have been modified with retention of a significant fraction of the initial activity. It seems likely that at least one of these groups (amino, carboxyl, tyrosinyl, tryptophanyl) should be in contact with the receptor in the toxin-receptor complex. If so, that should imply that it is possible to chemically modify such an interacting group; the interaction from the remaining groups should still be sufficient to bind the toxin to its target. There exists a great variety of cholinergica, which have only one obvious feature in common, a strongly positively charged group, which is suggested to be a recognition site, the function of which is to make it possible for the molecule to recognize the target. An invariant arginine residue is assumed to provide the recognition site in the neurotoxins.     

The complete primary structure of toxin CM-1b from Hemachatus haemachatus (Ringhals) snake venom

Toxin CM-1b was purified from Hemachatus haemachatus venom. The purified toxin contains 57 amino acids including 8 half-cystine residues in a single polypeptide chain. The complete primary structure of CM-1b has been established. The sequence and the invariant amino acid residues of CM-1b resemble those of the Naja-type toxins. In toxin CM-1b one of the structurally invariant amino acid residues proline 48, of the short neurotoxins has been replaced by a arginine, while it contains none of the functionally invariant amino acid residues.     

Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan banded krait (Bungarus multicinctus) venom

By ion exchange chromatography followed by gel filtration, a polypeptide toxin was purified from Formosan banded krait (Bungarus multicinctus) venom. The toxin had a molecular weight of 7000 ± 300 and showed an amino acid composition characteristic of cardiotoxin from cobra venom. The i.p. ld₅₀ value of the toxin was 2.5 (1.9–3.2) mg per kg mouse. Pharmacological studies showed that the toxin (10 μg/ml) induced contracture in chick and mouse skeletal muscles, depolarized the cell membrane of the mouse diaphragm, arrested the contraction of spontaneously beating atria and the electrically driven ventricle strip of the rat, and caused direct hemolysis of guinea-pig erythrocytes. From these chemical and pharmacological characterizations it was concluded that this toxin has characteristics similar to those of cobra venom cardiotoxins.     

Purification, characterization and cDNA cloning of two natterin-like toxins from the skin secretion of oriental catfish Plotosus lineatus

The oriental catfish Plotosus lineatus is known to contain proteinaceous toxins in the skin secretion as well as in the venom gland. However, detailed properties and primary structures of the skin toxins have not been clarified. In this study, two proteinaceous toxins (toxins I and II) were purified from the skin secretion of oriental catfish by a combination of gel filtration, anion-exchange HPLC and hydroxyapatite HPLC. Toxins I and II are monomeric simple proteins with almost the same molecular mass (35 kDa for toxin I and 37 kDa for toxin II) and are distinguishable from each other in isoelectric point (6.5 for toxin I and 5.1 for toxin II). Both toxins display lethal, edema-forming and nociceptive activities, although toxin I is significantly more potent than toxin II. The primary structures of toxins I and II were elucidated by cloning experiments based on the determined partial amino acid sequences. Toxins I (317 amino acid residues) and II (315 amino acid residues) share as high as 86% sequence identity with each other and are also highly homologous (56–75% identities) with the known fish natterin-like proteins.     

The isolation and purification of two peptides from the venom of Buthus martensii Karsch

Two peptides that extensively prolong action potentials (APs) in rat and frog nerves have been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. Action potentials recorded in the presence of nanomolar concentrations of the peptides were extensively prolonged without much attenuation in their heights. The N-terminal sequences of both the peptides, BMK 9(3)-1 and BMK 9(3)-2, were determined. The N-terminal sequences of BMK 9(3)-1 and BMK 9(3)-2 were found to be: GRDAYIADSEN-PYF-GANPN and GRDAYIADSEN-PYT-ALNP. Sequence similarity comparisons to other -scorpion toxins suggest that the two blanks in each of the sequences are cysteines. The first 20 residues of the two BMK peptides differ by only three amino acid substitutions. The molecular weight (MW) of BMK 9(3)-1 and BMK 9(3)-2 were determined by LC/MS/MS to be 7020 and 7037 Da. Since both of the peptides prolong APs when both K⁺ and Ca⁺⁺ channels are blocked and show sequence similarity to other -neurotoxins, it appears likely that BMK 9(3)-1 and BMK 9(3)-2 act to alter Na channel inactivation to produce their effects. The first 20 residues of BMK 9(3)-2 are identical to those observed for makatoxin I, a toxin isolated from Buthus martensii Karsch venom, that alters nitric oxide transmitter release. Since the two toxins also have very similar molecular weights, BMK 9(3)-2 may be identical to makatoxin I; however, BMK 9(3)-2 acts to alter Na channels to exert its effect, thus the two toxins may differ, or if they are identical, they can exert effects on both neural transmission and AP propagation.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector

The complete covalent structure of the insect toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a “protein” or a “peptide” program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after performic oxidation, and by partial labeling of the half cystine residues with [¹⁴C]-iodoacetic acid and determining the specific radioactivities of the S-[¹⁴C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys¹² in mammal toxin II is replaced by Cys³⁸ in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half-cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.