M. El Ayeb

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors.

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33‐mer basic peptide cross‐linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid‐phase method, and purified after folding/oxidation by using both C18 reversed‐phase and ion exchange high‐pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half‐cystine pairing pattern of sMCa was identified by enzyme‐based cleavage and Edman sequencing. The pairings were Cys3‐Cys17, Cys10‐Cys21, and Cys16‐Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD50, 20 μg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels.

A new toxin acting on K+ channels, maurotoxin (MTX), has been purified to homogeneity from the venom of the chactoid scorpion Scorpio maurus. MTX is a basic single chain 34 amino acid residue polypeptide, amidated at its C terminal, and crosslinked by four disulfide bridges. It shows 29–68% sequence identity with other K+ channel toxins, and presents an original disulfide pattern, the last two half‐cystine residues (31–34) being connected. Although the first three disulfide bonds have not been defined experimentally, modelling based on the structure of charybdotoxin favored two combinations out of six, one of which has two bridges (3–24 and 9–29) in common with the general motif of scorpion toxins. The last bridge would connect residues 13 and 19. MTX inhibits the binding to rat brain synaptosomal membranes of both [125I]apamin, a SKCa channel blocker (IC50 5 nM), and [125I]kaliotoxin, a Kv channel blocker (IC50 30 pM). MTX blocks the Kv1.1, Kv1.2 and Kv1.3 currents expressed in Xenopus oocytes with IC50 of 45, 0.8 and 180 nM, respectively. MTX represents a member of a new class of short toxins with 4 disulfide bridges, active on voltage‐dependent K+ channel and also competing with apamin for binding to its receptor.

SOLUTION STRUCTURE OF MAUROTOXIN, A SCORPION TOXIN FROM SCORPIO MAURUS, WITH HIGH AFFINITY FOR VOLTAGE-GATED POTASSIUM CHANNELS

Maurotoxin (MTX), purified from the scorpionid Scorpio maurus is a potent ligand for potassium channels. It shows a broad specificity as being active on Kv1.1 (Kd = 37 nM), Kv1.2 (Kd = 0.8 nM), Kv1.3 (Kd = 150 nM) voltage‐gated potassium channels, as well as on small‐conductance calcium‐activated potassium channels. It has a unique disulfide pairing among the scorpion toxins family. The solution structure of MTX has been determined by 2D‐NMR techniques, which led to the full description of its 3D conformation: a bended helix from residues 6 to 16 connected by a loop to a two‐stranded antiparallel β sheet (residues 23 to 26 and 28 to 31). The interaction of MTX with the pore region of the Kv1.2 potassium channel has been modeled according to their charge anisotropy. The structure of MTX is similar to other short scorpion toxins despite its peculiar disulfide pairing. Its interaction with the Kv1.2 channel involves a dipole moment, which guides and orients the toxin onto the pore, toward the binding site, and which thus is responsible for the specificity. Proteins 29:321–333, 1997.

In vivo protection against Androctonus australis hector scorpion toxin and venom by immunization with a synthetic analog of toxin II

A synthetic peptide mimicking the North African scorpion Androctonus australis hector toxin II was designed and produced by chemical solid-phase synthesis. It contains the entire sequence of toxin II (64 amino acid residues), with each half-cystine being replaced by the isosteric residue a-aminobutyric acid, and was thus devoid of disulfide bridges. This construct was totally nontoxic in mice even if large amounts, equivalent to 1000 times the LD50 of the original toxin, were injected by the intracerebroventricular route. The synthetic peptide, either as a monomer or polymerized by means of glutaraldehyde, induced the production of antitoxin neutralizing antibodies in immunized mice and rabbits. After three injections with either the monomeric or polymerized synthetic peptide, the immunized mice were protected against several lethal doses of the corresponding native toxin or scorpion venom. Six months after immunization, the mice were completely protected against challenge with eight LD50 of the original toxin. The protection was better when the polymerized synthetic peptide was used. One month after the start of the immunization program, it showed a good correlation between antibody titer and protection. However, antibody titer decreased with time but protection remained high. This suggests that additional factors other than circulating antibodies play a role in protective activity.

PURIFICATION AND CHARACTERIZATION OF A FIBRINOGENASE FROM VIPERA LEBETINA (DESERT ADDER) VENOM

A fibrinogenase from Vipera lebetina venom was isolated by gel filtration in a Superose 12 column prep grade HR 16/50 and by ion-exchange in a Mono Q HR 5/5 column. The purified enzyme, which was obtained with a yield of 8 mg from 60 mg of crude venom, is a glycoprotein having an isoelectric point of 5.9 +/- 0.1 and a mol. wt of 26,000 +/- 1000 as estimated by SDS-PAGE. The biochemical characterization of the enzyme revealed that it hydrolyzes readily the B beta chain of fibrinogen and the A alpha chain as well as fibrin and casein. Over a pH range from 4 to 11 the enzyme was not inactivated by a 20 min treatment at 90 degrees C. The isolated fibrinogenase is inhibited by ethylenediamine tetraacetic acid, dithiothreitol and L-cysteine but not by phenylmethylsulfonyl fluoride. On the other hand, it is activated by Ca2+ and Mg2+. Purified fibrinogenase up to a dose of 100 micrograms/mouse shows no toxicity and has no hemorrhagic activity.

Bot IT2, a toxin paralytic to insects from the Buthus occitanus tunetanus venom modifying the activity of insect sodium channels

The effects of insect toxin Bot IT2, purified from the venom of the scorpion Buthus occitanus tunetanus, were investigated on the isolated giant axon and on isolated dorsal unpaired median (DUM) neurone of the cockroach Periplaneta americana under current- and voltage-clamp conditions, using the double-oil-gap technique and the patch-clamp technique, respectively. In both preparations, Bot IT2, induces a limited depolarization together with the development of a repetitive activity in axon and an increase of spontaneous discharge frequency in DUM neurone. After artificial hyperpolarization to the normal resting level, plateau potentials can be evoked in both preparations. Under voltage-clamp conditions, Bot IT2 induces a similar effect in axon and DUM neurone by acting specifically on sodium channels. The peak sodium current is decreased and simultaneously a new current with very slow activation-deactivation kinetics is developed. Voltage dependence of this slow current is not very different from that of the control. The inactivation of the fast component is incomplete because it is masked by the development of the slow component. These results suggest that Bot IT2 modifies the kinetics of insect sodium channel activation, and the transformation of normal fast channels into slow ones is discussed.

Maurotoxin, a four disulfide bridges scorpion toxin acting on K+ channels

Maurotoxin, a toxin from the venom of the Tunisian chactoid scorpion Scorpio maurus, has been purified to homogeneity by gel filtration/reversed-phase HPLC, and characterized. It is a basic and C-terminal amidated 34-residue polypeptide cross-linked by four disulfide bridges. From Edman sequencing results, only six different pairings between the first six half-cystines were retained whereas a disulfide bridge was predicted between the two half-cystines in positions 31 and 34. Modelling based on the structure of charybdotoxin favored two different pairings, one of which possessed two disulfides in common with the general motif of scorpion toxins. The solid-phase technique was used to obtain synthetic maurotoxin, sMTX. The half-cystine pairings of sMTX were determined by enzymatic cleavage and were found to be Cys3 Cys24, Cys9-Cys29, Cys13-Cys19, and Cys31-34, in agreement with experimental data obtained with natural maurotoxin. Both natural and synthetic maurotoxins were lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). They blocked the Kv1.1, Kv1.2, and Kv1.3 channels expressed in Xenopus oocytes with almost identical half-effects (IC50) in the range of 40, 0.8 and 150 nM, respectively. They also competed with 125I-apamin (SKca channel blocker) and 125I-kaliotoxin (Kv channel blocker) for binding to rat brain synaptosomes with IC50 of about 5 and 0.03 nM. As the natural and synthetic maurotoxins exhibit indistinguishable physicochemical and pharmacological properties, they are likely to adopt the same half-cystine pairing pattern which is unique among known scorpion toxins. However, this disulfide organization is different from those reported for Pandinus imperator and Heterometrus spinnifer toxins 1 (Pi1 and HsTx1), two novel four-disulfide bridged K+ channel-acting scorpion toxin sharing about 50-70% sequence identity with maurotoxin.

Anti-platelet activity of the peptides composing the lebetin 1 family, a new class of inhibitors of platelet aggregation

We have purified from Vipera lebetina venom a family of inhibitors of platelet aggregation, named Lebetins. They are composed of two peptide groups of short (Lebetin 1: L1alpha: GDNKPPKKGPPNG; L1beta: DNKPPKKGPPNG) and long (Lebetin 2: L2alpha: GDNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG; L2beta: DNKPPKKGPPNGCFGHKIDRIGSHSGLGCNKVDDNKG) size. The sequence presenting anti-platelet activity is mainly present within the Lebetin 1 sequence [Barbouche, R. Marrakchi, N., Mansuelle, P., Krifi, M., Fenouillet, E., Rochat, H. and El Ayeb, M. (1996) Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization. FEBS Lett. 392, 6-10]. Here, the peptides that compose the Lebetin 1 family were synthesized. Their respective activity was determined. Synthetic L1alpha and L1beta inhibited collagen-induced platelet aggregation in the nanomolar range. A peptide corresponding to L1beta deleted by D at its N terminus (L1gamma) also inhibited platelet aggregation potently; further truncation of L1gamma impaired its activity. Because L1 peptides efficiently inhibited fibrinogen-induced alpha-chymotrypsin treated-platelet aggregation, we tested whether they act mainly through the inhibition of platelet binding to fibrinogen and showed that they failed to inhibit platelet binding to fibrinogen-coated wells. The activity of L1 peptides was also tested in vivo: their intravenous administration strongly inhibited collagen-induced thrombocytopenia in rats.

Lebetin peptides: potent platelet aggregation inhibitors.

Lebetins from Macrovipera lebetina snake venom constitute a new class of inhibitors of platelet aggregation. There are two groups of peptides: lebetin 1 (L1; 11- to 13-mer) and lebetin 2 (L2; 37- to 38-mer). The short lebetins are identical to the N-terminal segments of the longer ones. They inhibit platelet aggregation induced by various agonists (e.g. thrombin, PAF-acether or collagen). The shortest lebetin (11-mer) shows potent inhibition of rabbit (IC50 = 7 nM) and human (IC50 = 5 nM) platelets. They prevent collagen-induced thrombocytopenia in rats. N- and C-terminal-truncated synthetic L1γ (sL1γ; 11-mer) is less active in inhibiting platelet aggregation than the native peptide. Results from Ala scan studies of the sL1γ peptide indicated that replacement of the residues (P3, G7, P8, P9 or N10) resulted in a remarkable drop in the activity, whereas replacement of residues K2, P4 or K6 by Ala resulted in enhancement of the antiplatelet activity by at least 10-fold. To examine the activity of multimeric L1γ, several multimeric peptides were synthesized using the multiple-antigen peptide system assembled on a branched lysine core and their antiplatelet activity was evaluated in vitro. The largest multimeric peptides showed a 1,000-fold increase in antiplatelet activity.