Lourival D. Possani

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.

Peptides and genes coding for scorpion toxins that affect ion-channels.

Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.

Demonstration of central γ-aminobutyrate-containing nerve terminals by means of antibodies against glutamate decar☐ylase

Abstract A new procedure is described to obtain specific antibodies against mouse glutamate decar☐ylase using a purified glutamate decar☐ylase preparation obtained by affinity chromatography. Antibodies prepared against the Sepharose-bound glutamate decar☐ylase caused a concentration-dependent inhibition of glutamate decar☐ylase activity. Furthermore, the glutamate decar☐ylase antibodies gave a single precipitation band in double immunodiffusion, immunoelectrophoresis and in counterimmunoelectrophoresis analysis when reacted against crude extracts from mouse or rat brain. Immunohistochemical analysis in the rat brain demonstrated specific glutamate decar☐ylase-related immunofluorescence, presumably in γ-aminobutyrate-containing nerve terminals, but in no case in cell bodies in any part of the brain and the spinal cord. Two principal types of presumed γ-aminobutyrate-containing nerve terminals could be demonstrated. One type is strongly glutamate decar☐ylase immunoreactive, appears to have fairly large varicosities, may possibly make axosomatic and axodendritic contacts, and is confined mainly to the deep cerebellar nuclei, the nucleus vestibularis lateralis, the substantia nigra, the globus pallidus and the substantia innominata together with adjacent parts of the medial forebrain bundle and the dorsal part of the olfactory tubercle. In all these areas they are found in high densities. It is suggested, partly in agreement with previous findings that this type of γ-aminobutyrate-containing nerve terminal belongs to Golgi type I nerve cells (with long projections), contains high amounts of glutamate decar☐ylase enzyme protein and may mediate postsynaptic inhibition. The other type of γ-aminobutyrate-containing nerve terminal is weakly to moderately glutamate decar☐ylase immunoreactive, appears to have fine varicosities, may possibly make both axosomatic, axodendritic and axoaxonic contacts and is found all over the brain and the spinal cord in low to high densities. The distribution of this type correlates fairly well with the known distribution of glutamate decar☐ylase activity in the rat brain. It is suggested that this type of γ-aminobutyrate-containing nerve terminal mainly belongs to Golgi type II neurons (interneurons), contains relatively low amounts of glutamate decar☐ylase enzyme protein, and mediates both pre- and postsynaptic inhibition. The findings underline the existence of a new type of γ-aminobutyrate-containing Golgi type I neuron, controlling activity in the substantia innominata and in the outflow from Callejas islands of the olfactory tubercle and the view that γ-aminobutyrate may play a role in synaptic processes in all parts of the brain and the spinal cord.

Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom

A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti‐bacterial activity and a potent inhibitory effect on the ookinete (ED50 0.7 μM) and gamete (ED50 10 μM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N‐terminal segment and to some defensins in its C‐terminal region. Its gene was cloned from a cDNA library.

Scorpionism and serotherapy in Mexico.

In Mexico, scorpionism is an endemic public health problem. The exact number of human accidents is unknown, but partial statistics suggests numbers close to 200,000 per year. The documented number of fatality cases is in the order of 310 people per year. We currently use horse antiserum in patients who show a clear picture of intoxication. Our personal experience in treating 38,068 people, from which over 20,000 received serotherapy, shows that the antiserum is very effective, in that none of the patients died.

Novel interactions between K+ channels and scorpion toxins

K(+) channels are macromolecules embedded in biological membranes, where they play a key role in cellular excitability and signal transduction pathways. Knowledge of their structure should help improve our understanding of their function and lead to the design of therapeutic compounds. Most pharmacological and structural characteristics of these channels have been elucidated by using high-affinity channel blockers isolated from scorpion venoms. Recent data on the three-dimensional structures of K(+) channels and novel scorpion toxins suggest a variety of novel interacting modes of these channels and toxins, which should help increase our understanding of the K(+) channel structure-function relationship.

TRIABIN, A HIGHLY POTENT EXOSITE INHIBITOR OF THROMBIN

Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug. It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time. However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay. It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion. These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin. The protein was partially sequenced and the information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained. The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay. In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a K of about 3 pM.

The primary structure of noxiustoxin: A K+ channel blocking peptide, purified from the venom of the scorpion Centruroides noxius Hoffmann

Noxiustoxin, component II-11 from the venom of scorpion Centruroides noxiusHoffmann, was obtained in pure form after fractionation by Sephadex G-50 chromatography followed by ion exchange separation on carboxy-methylcellulose columns (17). The primary structure of Noxiustoxin, a polypeptide 39 amino acid residues long was determined by automaticEdman degradation and chemical cleavage with cyanogen bromide followed by amino acid analysis of the two resulting peptides. Its sequence is: Thr−Ile−Ile−Asn−Val−Lys−Cys−Thr−Ser−Pro−Lys−Gln−Cys−Ser−Lys−Pro−Cys−Lys−Glu−Leu−Tyr−Gly−Ser−Ser−Ala−Gly−Ala−Lys−Cys−Met−Asn−Gly−Lys−Cys−Lys−Cys−Tyr−Asx−Asn, with a molecular weight of 4,184±6. No histidine, arginine, tryptophan or phenylalanine was found. Noxiustoxin is the first short toxin directed against mammals and the first K+ channel blocking polypeptide-toxin (4) found in scorpion venoms.

Toxins and genes isolated from scorpions of the genus Tityus

Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.

Oxidative refolding chromatography: folding of the scorpion toxin Cn5

We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components—GroEL minichaperone (191–345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl–prolyl isomerase—were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66–residue toxin, which has four disulfide bridges and a cis peptidyl–proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.