Purification and partial characterization of a ‘short' insectotoxin‐like peptide from the venom of the scorpion <i>Parabuthus schlechteri</i>

Tytgat, Jan; Debont, Tom; Rostoll, Karin; Müller, G; Verdonck, Fons; Daenens, Paul; Walt, J. J. Van der; Possani, Lourival D.

doi:10.1016/s0014-5793(98)01589-0

Cited by 35 publications

(21 citation statements)

References 20 publications

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“…Recent studies have identified many low-molecular-mass peptide toxins from Lqh and other scorpion venoms. Sequence comparisons have determined that Lqh-8/6 from L. quinquestriatus hebraeus (2), Bs8 and Bs14 from Buthus sindicus (4), and PBITx1 from Parabuthus schlechteri (53) are similar to ClTx from Lqq venom. Therefore, there is a possibility that this Lqh toxin will be one previously identified; alternatively, this study will result in the identification of a novel peptide toxin that selectively inhibits CFTR.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of CFTR channels by a peptide toxin of scorpion venom

Fuller

Zhang²,

Cui³

et al. 2004

American Journal of Physiology-Cell Physiology

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Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified that interact with known anion-selective channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR channels are expressed in epithelial cells and are associated with several genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the Cl− permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered by the lack of a high-affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion Leiurus quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing single-channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or adenosine 5′-(β,γ-imido)triphosphate, and block was strengthened on reduction of extracellular Cl− concentration, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore-block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function.

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Section: Discussionmentioning

confidence: 99%

Inhibition of CFTR channels by a peptide toxin of scorpion venom

Fuller

Zhang²,

Cui³

et al. 2004

American Journal of Physiology-Cell Physiology

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“…Preliminary studies indicated that native PBTx1 blocks Shaker-type Kv1.1 channels expressed in Xenopus laevis oocytes with a K d value of ϳ150 nM (34). A more in-depth analysis has revealed the presence of a more potent contaminating peptide in this sample, PBTx3, recently characterized by our group (30).…”

Section: Physiological Effects Of the New Subfamily ␣-Ktx11mentioning

confidence: 85%

“…Five residues recognized as "critical residues" in Kv-blocking activity (11) in ChTx, Lys 27 , Met 29 , Asn 30 , Arg 34 , and Tyr 36 , are well conserved among different ␣-KTx peptides. However, none of them is conserved in PBTx1.…”

Section: Discussionmentioning

confidence: 99%

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A Subfamily of Acidic α-K+ Toxins

Huys

Olamendi-Portugal²,

García‐Gómez³

et al. 2004

Journal of Biological Chemistry

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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 ␣-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 ␣-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.

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“…A study was undertaken to find compounds or toxins in the venom of P. transvaalicus that modulate physiological processes at the cellular level; this was done for the following reasons: (a) very little is known about the bioactive substances present in the venom of this scorpion [7,8]; (b) the discovery of new toxins can be the key to gain insight into the molecular mechanisms of scorpionism; (c) selective toxins can be used for purifying channels from native tissue, determining their subunit composition [9] and for elucidating the pharmacology and physiological roles of voltagedependent Na + , Ca 2+ and K + channels [10][11][12] in target tissues. Voltage-dependent K + channels in particular serve important functions in many signal-transduction pathways in the nervous system: they are involved in neuron excitability; they influence the resting membrane potential, the waveforms and frequencies of action potentials; and they determine the thresholds of excitation [13].…”

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confidence: 99%

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

et al. 2002

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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 Ôa-KTx1.1Õ). PBTx3 is the tenth member (called Ôa-KTx1.10Õ) 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 (K d ) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 lM, 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 X a (fX a ) 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 threedimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel b sheet and a single a-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other a-KTx scorpion toxins.

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Purification and partial characterization of a ‘short' insectotoxin‐like peptide from the venom of the scorpion Parabuthus schlechteri

Cited by 35 publications

References 20 publications

Inhibition of CFTR channels by a peptide toxin of scorpion venom

Inhibition of CFTR channels by a peptide toxin of scorpion venom

A Subfamily of Acidic α-K+ Toxins

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

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