MeuTXKβ1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity

doi:10.1016/j.bbapap.2009.12.017

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Volume 1804, Issue 4, April 2010, Pages 872-883

https://doi.org/10.1016/j.bbapap.2009.12.017 Get rights and content

Abstract

Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKβ1) belonging to the TsTXKβ molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1–21 and 22–72 fragments (named N(1–21) and C(22–72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKβ1) exhibited a low affinity for K⁺ channels and cytolytic effects against bacteria and several eukaryotic cells. N(1–21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22–72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKβ1 presents a typical scorpion toxin scaffold in water and its α-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1–21) and an ab initio structure model of MeuTXKβ1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKβ-related peptides and antiparasitic scorpines which both comprise the βSPN (β-KTxs and scorpines) family.

Introduction

Scorpion toxins with cysteine-stabilized alpha/beta fold (CSαβ) target various ion channels of excitable membranes [1], [2], [3], [4], [5]. The short-chain scorpion toxins affecting K⁺ channels usually are composed of 23–42 amino acids with 3 or 4 disulfide bridges and are classified as α-KTx molecular subfamilies [6], most of which block voltage-gated K⁺ channels (Kv) by a dyad motif directly interacting with the channel pore [7], [8], [9]. However, other interacting modes have also been proposed to account for high-affinity binding to Kv channels for some toxins without the dyad [2], [10].

Besides these α-KTxs, venoms from scorpions belonging to Buthidae, Caraboctonidae and Scorpioninae contain β-KTxs of 61–75 amino acids [6]. Based on sequence similarity, these molecules can be further divided into three classes [11]: 1) class 1 contains TsTXKβ (TstβKTx), AaTXKβ, BmTXKβ2, TdiβKTx, and TcoβKTx [12], [13], [14], [15], of which only TsTXKβ (TstβKTx) has been characterized as a blocker of Kv channel; and 2) class 2 consists of BmTXKβ, HgeβKTx, TcoKIK, TdiKIK, and TtrKIK. Recombinant BmTXKβ is a blocker of transient outward K⁺ current (I_to) in rabbit atrial myocytes, which is fast-inactivating and associated with heteromultimeric channels with Kv4.2 and Kv4.3 subunits [16]. HgeβKTx exhibits strong cytolytic effects towards broad targets including bacteria (Bacillus subtilis and Staphylococcus aureus), human erythrocytes and frog oocytes; 3) class 3 includes some scorpine-related peptides (e.g. scorpine, opiscorpine1–4, HgeScplp1, HgeScplp2, and heteroscorpine1) [17], [18], [19], [20], [21], a group of antimicrobial defensins with sequence similarity to class 1. Whereas full-length scorpine and HgeScplp1 are typical defensins, the K⁺ channel-blocking effect of HgeScplp1 appears to reside in its C-terminal domain.

In this work, we report systematic characterization of a new TsTXKβ-related peptide from the scorpion Mesobuthus eupeus venom gland by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. rMeuTXKβ1 is an anti-Plasmodium peptide with diverse functional features including low affinity binding to K⁺ channels and toxic effects on bacteria and mouse erythrocytes. Structural and functional evidence for a common ancestral origin between TsTXKβ-related peptides and the antimicrobial scorpines has been uncovered.

Section snippets

Screening of cDNA library

The cDNA library of the M. eupeus venom gland constructed by a PCR-based approach has been described previously [22]. Clones containing an insert of 300–600 bp potentially encoding open reading frames (ORFs) of polypeptide precursors were selected for DNA sequencing. Primer T25V (5′-TTTTTTTTTTTTTTTTTTT TTTTTTA/G/C-3′) was used for random sequencing of positive clones. Nucleotide sequences of MeuTXKβ1 and MeuTXKβ2 have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) under

Molecular and biochemical characterization of MeuTXKβ1

From the cDNA library of the venom gland of M. eupeus, we isolated and identified two clones (BeL-62 and BeL-226) coding for two peptide precursors with only one amino acid substitution. Fig. 1A shows the nucleotide and deduced amino acid sequences of these two cDNAs which are 491 bp containing an ORF encoding a peptide of 91 amino acid in length. The deduced amino acid sequence starts with a 19-residue signal peptide and ends with a 72-residue mature peptide. These two peptides are named

Discussion

The present study reports molecular cloning, biochemical purification, recombinant expression, chemical refolding and structural/functional characterization of a new scorpion long-chain K⁺ channel toxin-like peptide MeuTXKβ1. As a new anti-Plasmodium peptide, rMeuTXKβ1 exhibited diverse cytolytic activities. Apart from the inhibition of the parasite P. berghei, it can bind K⁺ channels on rat brain synaptosomes, and lyse the bacterium and eukaryotic cells (oocytes and mouse erythrocytes).

Conclusion

In this work, we extended the functional spectrum of the two-domain TsTXKβ molecular subfamily and further clarified its structural features. rMeuTXKβ1 is the first TsTXKβ-related peptide characterized to have cytolytic effect against bacteria, oocytes, mouse erythrocytes and parasites, which supports close relationship between scorpion venom-derived TsTXKβ-related peptides and antimicrobial scorpine-related peptides. Successful expression of rMeuTXKβ1 in E. coli lays the basis for further

Acknowledgements

This work was supported by the following grants: 1) the National Natural Science Foundation of China (30730015 and 30621003); 2) the 973 Program from the Ministry of Science and Technology of China (2010CB945304); 3) Bilateral Cooperation for the 16th Session of the Sino-Belgian S&T Mixed Commission to S. Z; and 4) OT/05/64, FWO-Vlaanderen (G.0330.06 and G.0257.08), BIL 07/10 (China) to J.T.

References (40)

R.C. Rodríguez de la Vega et al.
Novel interactions between K⁺ channels and scorpion toxins
Trends Pharmacol. Sci.
(2003)
M.F. Martin-Eauclaire et al.
New “Birtoxin analogs” from Androctonus australis venom
Biochem. Biophys. Res. Commun.
(2005)
J. Tytgat et al.
A unified nomenclature for short-chain peptides isolated from scorpion venoms: α-KTx molecular subfamilies
Trends Pharmacol.
(1999)
M. Dauplais et al.
On the convergent evolution of animal toxins
J. Biol. Chem.
(1997)
K.N. Srinivasan et al.
κ-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function: importance of the functional diad in potassium channel selectivity
J. Biol. Chem.
(2002)
K.M. Giangiacomo et al.
Molecular basis of alpha-KTx specificity
Toxicon
(2004)
E. Diego-García et al.
Wide phylogenetic distribution of Scorpine and long-chain beta-KTx-like peptides in scorpion venoms: identification of “orphan” components
Peptides
(2007)
C. Legros et al.
Evidence for a new class of scorpion toxins active against K⁺ channels
FEBS Lett.
(1998)
S. Zhu et al.
Molecular cloning and sequencing of two ‘short chain’ and two ‘long chain’ K⁺ channel-blocking peptides from the Chinese scorpion Buthus martensii Karsch
FEBS Lett.
(1999)
Z. Cao et al.
Expression, purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta
Peptides
(2003)

R. Conde et al.

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

FEBS Lett.

(2000)

N. Uawonggul et al.

Purification and characterization of Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom

Toxicon

(2007)

E.F. Schwartz et al.

Mass spectrometry analysis, amino acid sequence and biological activity of venom components from the Brazilian scorpion Opisthacanthus cayaporum

Toxicon

(2008)

S. Zhu et al.

Molecular characterization of a new scorpion venom lipolysis activating peptide: evidence for disulfide bridge-mediated functional switch of peptides

FEBS Lett.

(2006)

Y. Yuan et al.

Functional expression of a Drosophila antifungal peptide in Escherichia coli

Protein Expres. Purif.

(2007)

R. Romi et al.

Synthesis and characterization of kaliotoxin

J. Biol. Chem.

(1993)

P. Guntert et al.

Torsion angle dynamics for NMR structure calculation with the new program DYANA

J. Mol. Biol.

(1997)

M.C. Rodríguez et al.

Effect of a cecropin-like synthetic peptide (Shiva-3) on the sporogonic development of Plasmodium berghei

Exp. Parasitol.

(1995)

F.M. Marassi et al.

Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy

Biophys. J.

(1999)

C. Legros et al.

Influence of a NH₂-terminal extension on the activity of KTX₂, a K⁺ channel blocker purified from Androctonus australis scorpion venom

FEBS Lett.

(1997)

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Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spectrometry and venomic analysis confirmed the presence of elastase-like, cathepsin-O-like proteases and a neprilysin-like metalloproteinase. We did not detect phospholipase A₂, C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a β-KTx-like peptide, and other putative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic effects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the effect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological effects at 24 h, which include myonecrosis, weak hemorrhage, and inflammatory infiltration. We observed an apparent thrombotic effect in the skin blood vessels, but no in vitro fibrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of time.
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Scorpion venom is an unmatched source of selective high-affinity ligands of potassium channels. There is a high demand for such compounds to identify and manipulate the activity of particular channel isoforms. The objective of this study was to obtain and characterize a specific ligand of voltage-gated potassium channel K_V1.2. As a result, we report the remarkable selectivity of the peptide MeKTx11-1 (α-KTx 1.16) from Mesobuthus eupeus scorpion venom to this channel isoform. MeKTx11-1 is a high-affinity blocker of K_V1.2 (IC₅₀ ∼0.2 nM), while its activity against K_V1.1, K_V1.3, and K_V1.6 is 10 000, 330 and 45 000 fold lower, respectively, as measured using the voltage-clamp technique on mammalian channels expressed in Xenopus oocytes. Two substitutions, G9V and P37S, convert MeKTx11-1 to its natural analog MeKTx11-3 (α-KTx 1.17) having 15 times lower activity and reduced selectivity to K_V1.2. We produced MeKTx11-1 and MeKTx11-3 as well as their mutants MeKTx11-1(G9V) and MeKTx11-1(P37S) recombinantly and demonstrated that point mutations provide an intermediate effect on selectivity. Key structural elements that explain MeKTx11-1 specificity were identified by molecular modeling of the toxin–channel complexes. Confirming our molecular modeling predictions, site-directed transfer of these elements from the pore region of K_V1.2 to K_V1.3 resulted in the enhanced sensitivity of mutant K_V1.3 channels to MeKTx11-1. We conclude that MeKTx11-1 may be used as a selective tool in neurobiology.

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MeuTXKβ1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity

Abstract

Introduction

Section snippets

Screening of cDNA library

Molecular and biochemical characterization of MeuTXKβ1

Discussion

Conclusion

Acknowledgements

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Protein Expres. Purif.

J. Biol. Chem.

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