Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands.

A phospholipase A2, Laticauda colubrina phospholipase A2 II (LcPLA-II), and a phospholipase A2 homologue, Laticauda colubrina phospholipase A2 homologue I (LcPLH-I), were isolated from the venom of the yellow-lipped sea snake, Laticauda colubrina, from the Solomon Islands. LcPLA-II showed phospholipase A2 activity towards egg-yolk phosphatidylcholine (24 mumol/min per mg at optimal conditions at 37 degrees C) and lethal potency (LD50 45 micrograms/kg body wt. intravenously in mice). Both of the activities were lost by treatment with p-bromophenacyl bromide. LcPLH-I showed neither phospholipase A2 activity nor lethal potency at a dose of 4.5 mg/kg body wt. in mice. It was not modified by the treatment with p-bromophenacyl bromide. LcPLA-II and LcPLH-I bound Ca2+ at a 1:1 molar ratio with KCa values of 105 microM and 44 microM at pH 8.0 respectively. Elucidation of the amino acid sequences of these two proteins showed that each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. The two sequences are different from each other at 22 residues and highly homologous to those from other sources. The essential histidine residue for the phospholipase A2 activity at position 48 is replaced by an asparagine residue in the homologue LcPLH-I. Details of the separation of the peptides obtained by proteinase digestions of LcPLA-II and LcPLA-I and the determination of their amino acid sequences are given in Supplementary Publication SUP 50145 (14 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

Properties and biosynthesis of a neurotoxic protein of the venoms of sea snakes Laticauda laticaudata and Laticauda colubrina

1. A neurotoxic protein similar to erabutoxins a and b of Laticauda semifasciata was isolated in crystalline form from the venoms of Laticauda laticaudata and Laticauda colubrina. The name ;laticotoxin a' is proposed. 2. Laticotoxin a is homogeneous by CM-cellulose column chromatography, disc electrophoresis and ultracentrifugation and by terminal amino acid analyses. 3. Laticotoxin a consists of 62 amino acid residues. The molecular weight by ultracentrifuging is 6520. 4. The minimal 50% lethal dose of laticotoxin a by intramuscular injection to mice is 0.13mug./g. body wt. The toxin attacks the postsynaptic membrane, competing with acetylcholine. 5. Radioactive amino acids are incorporated into laticotoxin a in vivo. The incorporation is inhibited by puromycin, suggesting that the biosynthesis of the toxin follows the mechanism of protein biosynthesis, although the toxin molecule is rather small as a protein.     

The isolation of an easily reversible postsynaptic toxin from the venom of a sea snake, Laticauda semifasciata

A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10-20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate-polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD(50) of component LsIII by intramuscular injection was 1.24mug/g body wt. for mice and 0.45mug/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.     

Role of C-terminal tail of long neurotoxins from snake venoms in molecular conformation and acetylcholine receptor binding: proton nuclear magnetic resonance and competition binding studies

The role of the "C-terminal tail" segment of long neurotoxins has been investigated. The C-terminal four to five residues of alpha-bungarotoxin and Laticauda colubrina b have been cleaved off by carboxypeptidase P. The effect of such deletion on the toxin conformation has been monitored in proton nuclear magnetic resonance spectra and circular dichroism spectra. The removal of the C-terminal residues primarily affects the chemical shifts of proton resonances of the residues close to the cleavage site and does not induce a major conformational change. Therefore, the C-terminal tail of long neurotoxins does not appear to be important in maintaining the specific polypeptide chain folding. On the other hand, competition binding with tritium-labeled toxin alpha to Narke japonica acetylcholine receptor has revealed that cleavage of the C-terminal residues reduces the binding activity of alpha-bungarotoxin or Laticauda colubrina b to acetylcholine receptor. Thus it is likely that (the basic amino acid residues in) the C-terminal tail is directly involved in the binding of long neurotoxins to electric organ (and muscle) acetylcholine receptor.     

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.     

Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini.

From the venom of a sea snake Astrotia stokesii three neurotoxic components, toxins Astrotia stokesii a, b and c were isolated in 40, 15 and 5% yield by weight respectively of the whole venom. Their LD50 values for 20g mice were 0.13, 0.096 and 0.098 microgram/g body wt. respectively and accounted for almost all the lethal activity of the venom. Their amino acid sequences were determined. Astrotia stokesii a was composed of 60 amino acid residues with nine half-cystine residues and was quite homologous to other sea-snake short-chain neurotoxins in its amino acid sequence. Toxins Astrotia stokesii b and c were composed of 70 and 72 amino acid residues respectively with 10 half-cystine residues. They are the first long-chain neurotoxins with high activity isolated from sea-snake venoms. The C-terminal carboxy groups of toxins b and c were found to be amidated; the amidation is known for some polypeptides, but is novel for a protein. The amide group may make a hydrogen-bond with glutamic acid-39, which replaces a lysine that has so far been found invariably in long-chain neutrotoxins. Astrotia stokesii b and c are also novel in having phenylalanine-25 and isoleucine- or valine-42. The ordinary Tyr-Glu pair, which is observed in X-ray structure [Low, Preston, Sato, Rosen, Searl, Rudko & Richardson (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2991-2994] and n.m.r.study [Inagaki, Tatsumi, Miyazawa, Hori & Tamiya (1977) Abstr. Int. Congr. Pure Appl. Chem. 26th, p. 336] on erabutoxins may be replaced by a hydrophobic pair. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 5009o (30 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B1, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

The primary structure of the toxin Laticauda semifasciata III, a weak and reversibly acting neurotoxin from the venom of a sea snake, Laticauda semifasciata

A weak and reversibly acting neurotoxic protein of Laticauda semifasciata venom, Laticauda semifasciata III (component LsIII), was sequenced. Component LsIII consists of 66 amino acid residues and has five disulphide bridges, one of which was located between residues 26 and 30. The weak and reversible neurotoxicity of component LsIII is discussed in relation to its structure, which falls between those of the neuro- and cardiotoxins of sea snakes and Elapidae snakes isolated and sequenced so far.     

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.     

The isolation, properties and amino acid sequence of erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata

Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semifasciata, was isolated in pure form by repeated column chromatography on CM-cellulose columns. The toxin was crystallizable and monodisperse in rechromatography, disc electrophoresis and isoelectric focusing (isoelectric point, pH9.23-9.25). The molecular weight of the toxin, as estimated by gel filtration, was 7000. The toxin showed the same lethal activity to mice (0.13mug/g body wt., intramuscular injection) and the same effect on isolated frog muscle as erabutoxins a and b, the main toxic components of the venom. The toxin inhibited the acetylcholine contracture but not the potassium chloride contracture of muscle. Erabutoxin c consisted of 62 amino acid residues, containing one fewer lysine and one more histidine than erabutoxin a and one fewer lysine and one more aspartic acid (or asparagine) than erabutoxin b. Erabutoxin c was reduced, S-carboxymethylated and hydrolysed with trypsin. The only fragment different from the corresponding fragments from erabutoxin b was hydrolysed further with pepsin. One of the peptic fragments, which was assumed to have the aspartic acid (or asparagine) residue in question at the C-terminal end, was treated with carboxypeptidase A. The C-terminal residue was found to be an asparagine. It was therefore concluded that erabutoxin c was [51-asparagine]-erabutoxin b.     

The neurotoxins of the sea snake Laticauda schistorhynchus.

Erabutoxins a and b, the major neurotoxins in the venom of the sea snake Laticauda semifasciata, were detected in the venom of Laticauda schistorhynchus. The identity of the toxins was confirmed on the basis of elution position on CM-cellulose column chromatography, disc electrophoretic mobility, amino acid analysis and toxicity measurement.     

Primary structure of a short toxin from the venom of a vietnamese scorpion (Buthus sp.)

The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with sequences of short scorpion toxins led us to conclude that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.     

Studies on sea-snake venoms. Crystallization of erabutoxins a and b from Laticauda semifasciata venom

1. The toxic principles in the venom of the sea-snake Laticauda semifasciata were separated into two components by CM-cellulose chromatography and obtained in crystalline forms. They were named ;erabutoxins a and b'. 2. The homogeneity of each toxin was shown by rechromatography, by disk electrophoresis, by ultracentrifuging, by toxicity measurements before and after repeated crystallizations and by N-terminal analysis. 3. They had molecular weights of about 7000. Both of them contained 61 (or 62) amino acid residues/molecule. The only difference between erabutoxins a and b was that one of the aspartic acid (or asparagine) residues in erabutoxin a was replaced by a histidine residue in erabutoxin b. 4. Both of the toxins had LD(50) values of 0.15mug./g. body wt. for mice and 0.07mug./g. for rats. It was shown with frog-muscle preparations that they acted on postsynaptic membrane to block neuromuscular transmission.     

Primary Structure of a Short Toxin from the Venom of a Vietnamese Scorpion (Buthus sp.)

The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with known sequences of short scorpion toxins led to the conclusion that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.     

Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Snake venom toxins. The amino-acid sequences of three toxins (9B, 11 and 12A) from Hemachatus haemachatus (Ringhals) venom.

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group.     

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.     

Snake venom toxins. The amino acid sequence of three toxins (CM-2e, CM-4a and CM-) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-2e, CM-4a and CM-7) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex and by ion-exchange chromatography on CM-cellulose. They comprise 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of the three toxins have been elucidated. The toxicities, the serological properties, the sequences and the invariant amino acid residues of toxin CM-2e, CM-4a and CM-7 resemble the corresponding properties of the cytotoxin group.