Heterologous expression of a gene that codes for Pg8, a scorpion toxin of Parabuthus granulatus, capable of generating protecting antibodies in mice.

A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25ml of the anti-serum produced was capable of protecting up to 3 LD(50) doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.

A subfamily of acidic alpha-K(+) toxins.

Three homologous acidic peptides have been isolated from the venom of three different Parabuthus scorpion species, P. transvaalicus, P. villosus, and P. granulatus. Analysis of the primary sequences reveals that they structurally belong to subfamily 11 of short chain alpha-K(+)-blocking peptides (Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) Trends Pharmacol. Sci. 20, 444-447). These toxins are 36-37 amino acids in length and have six aligned cysteine residues, but they differ substantially from the other alpha-K(+) toxins because of the absence of the critical Lys(27) and their total overall negative charge. Parabutoxin 1 (PBTx1), which has been expressed by recombinant methods, has been submitted to functional characterization. Despite the lack of the Lys(27), this toxin blocks several Kv1-type channels heterologously expressed in Xenopus oocytes but with low affinities (micromolar range). Because a relationship between the biological activity and the acidic residue substitutions may exist, we set out to elucidate the relative impact of the acidic character of the toxin and the lack of the critical Lys(27) on the weak activity of PBTx1 toward Kv1 channels. To achieve this, a specific mutant named rPBTx1 T24F/V26K was made recombinantly and fully characterized on Kv1-type channels heterologously expressed in Xenopus oocytes. Analysis of rPBTx1 T24F/V26K displaying an affinity toward Kv1.2 and Kv1.3 channels in the nanomolar range shows the importance of the functional dyad above the acidic character of this toxin.

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction.

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.

Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels.

This report describes the isolation, primary structure determination, and functional characterization of two similar toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively). KLII from P. granulatus is identical to kurtoxin from Parabuthus transvaalicus (a 63 amino-acid long toxin) whereas KLI is a new peptide containing 62 amino acid residues closely packed by four disulfide bridges with a molecular mass of 7244. Functional assays showed that both toxins, KLI and kurtoxin from P. granulatus, potently inhibit native voltage-gated T-type Ca(2+) channel activity in mouse male germ cells. In addition, KLI was shown to significantly affect the gating mechanisms of recombinant Na(+) channels and weakly block alpha(1)3.3Ca(V) channels expressed in Xenopus oocytes. KLI and kurtoxin from P. granulatus represent new probes to study the role of ion channels in germ cells, as well as in cardiac and neural tissue.

Cationic peptides from scorpion venom can stimulate and inhibit polymorphonuclear granulocytes.

We have isolated two cationic peptides, sharing partial homology with each other, from the venom of South African scorpions. Both synthetic peptides-one containing 44 amino acids, the other containing 45 amino acids-were constructed. At submicromolar concentrations they can activate granulocytes as evidenced by a concentration dependent chemotaxis and exocytosis. They also strongly inhibit the production of superoxide anions. At higher concentrations they act as pore formers and induce leakage of the cells. These different effects may be related to their amphipathic structure.

Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa.

Two novel pore-forming peptides have been isolated from the venom of the South-African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of alpha-helical, cationic peptides. For the first time, a comparison of the primary structures of alpha-helical pore-forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40-50 amino acids contain a typical scorpion conserved sequence S(x)3KxWxS(x)5L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a pore-forming peptide previously isolated from the venom of the South-African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell-selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram-negative bacteria (1.3-25 micro m), while melittin and mastoparan, two well-known cytolytic peptides, were more effective against Gram-positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.

Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.

A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.

Determination of species-specific components in the venom of Parabuthus scorpions from southern Africa using matrix-assisted laser desorption time-of-flight mass spectrometry.

The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Parabuthus (Pocock 1890) by means of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) and to construct a species-specific venom code for species identification. The venom compositions of sixteen Parabuthus species, occurring in southern Africa, were characterized using representative peaks in the molecular mass range of 6400-8400 Da. This mass range is characteristic for the typical long-chain neurotoxins influencing sodium channels. Only a few of these peptides have been sequenced up to now. The impetus for development of these species-specific profiles was the observation of unique, highly reproducible mass spectral peaks within a specific species. An identification label for all the different species could be found using a minimum number of peaks. MALDI-TOFMS is therefore proposed as a complementary method to morphological and behavioural characteristics for species and ultimately subspecies discrimination.