Marie-France Martin-Eauclaire

A UNIFIED NOMENCLATURE FOR SHORT-CHAIN PEPTIDES ISOLATED FROM SCORPION VENOMS : ALPHA -KTX MOLECULAR SUBFAMILIES

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K+-channel-blocking peptides belonging to family 2 peptides and comprising 30-40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named alpha-KTx1-12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

A structural link between inactivation and block of a K+ channel.

Gating the ion-permeation pathway in K+ channels requires conformational changes in activation and inactivation gates. Here we have investigated the structural alterations associated with pH-dependent inactivation gating of the KcsA-Kv1.3 K+ channel using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. Transition of the KcsA-Kv1.3 K+ channel from a closed state at pH 7.5 to an inactivated state at pH 4.0 revealed distinct structural changes within the pore, correlated with activation-gate opening and inactivation-gate closing. In the inactivated K+ channel, the selectivity filter adopts a nonconductive structure that was also induced by binding of a pore-blocking tetraphenylporphyrin derivative. The results establish a structural link between inactivation and block of a K+ channel in a membrane setting.

Evidence for a new class of scorpion toxins active against K+ channels

cDNAs encoding novel long‐chain scorpion toxins (64 amino acid residues, including only six cysteines) were isolated from cDNA libraries produced from the venom glands of the scorpions Androctonus australis from Old World and Tityus serrulatus from New World. The encoded peptides were very similar to a recently identified toxin from T. serrulatus, which is active against the voltage‐sensitive ‘delayed‐rectifier potassium channel, but they were completely different from the long‐chain and short‐chain scorpion toxins already characterised. However, there was some sequence similarity (42%) between these new toxins, Aa TX Kβ and Ts TX Kβ, and scorpion defensins purified from the hemolymph of Buthidae scorpions Leiurus quinquestriatus and A. australis. Thus, according to a multiple sequence alignment using CLUSTAL, these new toxins seem to be related to the scorpion defensins.

Characterization of a new leiurotoxin I-like scorpion toxin: PO5 from Androctonus mauretanicus mauretanicus

Three novel peptide inhibitors of the SKCa channels were purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus using one step of RP‐HPLC and competition assays with [125I]apamin to rat brain synaptosomes. POi, PO2 and PO5 have K 0.5 of 100,100 and 0.02 nM, respectively, for the apamin binding site. The sequence of PO5 was established and compared to that of other scorpion toxins active on K + channels: it contains 31 residues and has a free carboxyl end. It shares sequence similarity with apamin and leiurotoxin I.

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main β-neurotoxin from the venom of the South American scorpion Tityus serrulatus

A cDNA encoding the main Tityus serrulatus β‐neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues: (ii) the amino acid sequence of the mature toxin: and (iii) an extra Gly‐Lys‐Lys tail at the C‐terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post‐translational cleavage by a carboxypeptidase specific for basic residues and the action of an α‐amidating enzyme. These results also show that the biosynthetic pathway for β‐toxins of ‘New World’ scorpion venoms is similar to that already described for α‐toxins of ‘Old World’ scorpion venoms.

Biochemical, pharmacological and genomic characterisation of Ts IV, an α‐toxin from the venom of the South American scorpion Tityus serrulatus

The venom of the scorpion, Tityus serrulatus, was fractionated to investigate the chemical and pharmacological properties of its α‐toxin content. Three α‐toxins (Ts III, Ts IV and Ts V) were purified by conventional chromatography (gel filtration and ion‐exchange chromatography), followed by immunoaffinity chromatography. Competition experiments using reference α‐ and β‐toxins suggested that these α‐toxins were contaminated with around 0.01% of β‐toxin. The sequence of the first 30 amino acids of Ts IV was established. Using an oligonucleotide probe, a cDNA encoding its precursor was cloned from a venom gland cDNA library. The primary structure deduced from the cDNA nucleotide sequence provides possible explanations for the polymorphism of these three molecules.

Scorpion α‐like toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage‐gated sodium channels in mammals and insects

α‐Like toxins, a unique group designated among the scorpion α‐toxin class that inhibit sodium channel inactivation, are highly toxic to mice but do not compete for α‐toxin binding to receptor site 3 on rat brain sodium channels. We analysed the sequence of a new α‐like toxin, which was also highly active on insects, and studied its action and binding on both mammalian and insect sodium channels. Action of the α‐like toxin on isolated cockroach axon is similar to that of an α‐toxin, and the radioactive toxin binds with a high affinity to insect sodium channels. Other sodium channel neurotoxins interact competitively or allosterically with the insect α‐like toxin receptor site, similarly to α‐toxins, suggesting that the α‐like toxin receptor site is closely related to receptor site 3. Conversely, on rat brain sodium channels, specific binding of 125I‐α‐like toxin could not be detected, although at high concentration it inhibits sodium current inactivation on rat brain sodium channels. The difficulty in measuring binding to rat brain channels may be attributed to low‐affinity binding due to the acidic properties of the α‐like toxins that also impair the interaction with receptor site 3. The results suggest that α‐like toxins bind to a distinct receptor site on sodium channels that is differentially related to receptor site 3 on mammalian and insect sodium channels.

Characterization of a new peptide from Tityus serrulatus scorpion venom which is a ligand of the apamin-binding site

A new ligand (Ts κ) of the apamin binding site on rat brain synaptosomes (K 0.5 = 300 pM) was purified and characterized from the venom of Tityus serrulatus. It is a polypeptide toxin of 35 amino acid residues, with three disulfide bridges. Its cDNA was amplified from a venom gland cDNA library and the nucleotide sequence determined. A model of Ts κ was constructed by amino acid replacement using charybdotoxin structure as determined by 1H nuclear magnetic resonance as starting model.

Structure-activity studies on scorpion toxins that block potassium channels

Scorpion venoms contain toxins that block different types of potassium channels. Some of these toxins have affinity for high conductance Ca(2+)-activated K+ channels and for dendrotoxin-sensitive voltage-dependent K+ channels. The structural features that determine the specificity of binding to different channel types are not known. We investigated this using natural and synthetic scorpion toxins. We have tested the effects of charybdotoxin (CTX) and two homologues (Lqh 15-1 and Lqh 18-2), iberiotoxin (IbTX), and kaliotoxin (KTX) from the scorpions Leiurus quinquestriatus hebreus, Buthus tamulus and Androctonus mauretanicus mauretanicus, respectively, and synthetic variants of CTX, namely CTX2-37, CTX3-37, CTX4-37, and CTX7-37, on a Ca(2+)-activated K+ current (IK-Ca) at a mammalian motor nerve terminal, and on the binding of a radiolabelled dendrotoxin, 125I-DpI, to voltage-dependent K+ channels on rat brain synaptosomal membranes. The native toxins contain 37-38 amino acid residues, they are over 30% identical in sequence (CTX and IbTX are 68% identical), and they have similar three-dimensional conformations. All toxins, except IbTX, displaced 125I-DpI from its synaptosomal binding sites: Lqh 18-2 (Ki = 0.25 nM), KTX (Ki = 2.1 nM), CTX (Ki = 3.8 nM), CTX2-37, (Ki = 30 nM), Lqg 15-1 (Ki = 50 nM), CTX3-37 (Ki = 60 nM), CTX4-37 (Ki = 50 nM), CTX7-37 (Ki = 105 nM). IbTX had no effect at 3 microM. When variants of CTX with deletions at the N-terminal portion were tested for their activity on IK-Ca on motor nerve terminals in mouse triangularis sterni nerve-muscle preparations, CTX3-37 and CTX4-37 were ineffective at 100 nM; and CTX7-37 was ineffective at up to 1 microM. IbTX and CTX (100 nM) completely blocked IK-Ca, but KTX (100 nM) did not affect the nerve terminal IK-Ca. Different residues appear to be important for interactions of the toxins with different K+ channels. IbTX did not displace dendrotoxin binding, but it did block IK-Ca, whereas KTX was as active as CTX against dendrotoxin binding but it did not affect the IK-Ca of the motor nerve terminals. The N-terminal section of the toxins appears to be particularly involved in block of IK-Ca at the motor nerve terminal: it is truncated in the inactive synthetic CTX variants; and it is positively charged at lysine-6 in KTX (which is inactive), but negatively charged in IbTX and neutral in CTX.(ABSTRACT TRUNCATED AT 400 WORDS)

A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.