Expression of an Insect Excitatory Toxin, BmK IT, from the Scorpion, Buthus martensii Karsch, and its Biological Activity

Hao, Chan-juan; Xu, Chenggang; Wang, Wei; Chai, Baofeng; Liang, Aihua

doi:10.1007/s10529-005-3905-0

Cited by 15 publications

(8 citation statements)

References 11 publications

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“…Given their abundance in crude venom and variation across species, the ␣D-conotoxins appear to have evolved as an alternative strategy for prey capture for species lacking ␣-, ␣A-, ␣S-, and -conotoxins. Whereas chemical synthesis of these proteins is expected to be challenging, recombinant production of other dimeric toxins of comparable size has been achieved (49,(62)(63)(64)(65)(66)(67). Co-crystallization studies with AChBP are expected to more precisely define the structure of ␣D-conotoxins and to establish the molecular basis of their binding interaction at the nAChR.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Class of Nicotinic Receptor Antagonists

Loughnan

Nicke

Jones

et al. 2006

Journal of Biological Chemistry

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The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, ␣D-VxXIIA, ␣D-VxXIIB, and ␣D-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an ϳ11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that ␣D-VxXIIA, ␣D-VxXIIB, and ␣D-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for ␣7 and ␤2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs. Nicotinic acetylcholine receptors (nAChRs)4 belong to the Cys-loop superfamily of pentameric ligand-gated ion channels. Neuronal nAChRs are generally formed from a combination of ␣ and ␤ subunits (␣2-␣10 and ␤2-␤4) that can assemble into a diversity of nAChR subtypes with different pharmacological and functional properties (1). Given their physiological importance, nAChRs are often targeted by venom peptides (2).Our current knowledge of the structure and function of nAChRs owes much to studies using snake toxins as biochemical or pharmacological tools to isolate and characterize this receptor (3). The "bungarotoxin" family of proteins in the venom of elapid and hydrophid snakes includes the -and ␣-neurotoxins, muscarinic toxins, cytotoxins, cardiotoxins, fasciculins, calciseptins, and mambins (4, 5). The structurally related short-chain and long-chain ␣-neurotoxins and the "weak non-conventional" snake toxins are comprised of 60 -74 amino acids, including 8 or 10 cystine residues and their structures have a three-fingered fold. The -and ␣-neurotoxins both inhibit nAChRs but differ in their specificity and binding kinetics (4).Conotoxins are small disulfide-rich peptide toxins found in the venom of predatory marine snails from the genus Conus. These venom peptides generally target a variety of voltage-gated and ligand-gated ion channels (6, 7). Conotoxins acting at nAChRs include the ␣-conotoxin, ␣A-conotoxin, -conotoxin, and ␣S-conotoxin families. The ␣-conotoxins are a large family of well characterized competitive nAChR antagonists with diverse subtype selectivities that allow the pharmacological dissection of nAChR subtypes (1,8). The structurally different ␣A-conotoxins are competitive antagonists with specificity for muscle nAChRs but lack the selectivity for the ␣1/␦ interface that is ex...

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Section: Discussionmentioning

confidence: 99%

Identification of a Novel Class of Nicotinic Receptor Antagonists

Loughnan

Nicke

Jones

et al. 2006

Journal of Biological Chemistry

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“…Scorpion neurotoxins have been most commonly expressed in Escherichia coli (Hao et al 2005). Nonetheless, neither the quantity nor the quality of the product is satisfactory (Pang et al 1992).…”

Section: Fusion Expression and Purification Of Bmatx14mentioning

confidence: 99%

Functional analysis of the α-neurotoxin, BmαTX14, derived from the Chinese scorpion, Buthus martensii Karsch

et al. 2006

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The gene encoding the BmalphaTX14 (alpha-neurotoxin TX14) protein, derived from the cDNA library of the Chinese scorpion Buthus martensii Karsch, was expressed in Pichia pastoris. The recombinant protein was purified by metal chelate affinity chromatography and gel filtration chromatography. Using patch-clamp technique, electrophysiological activity of rBmalphaTX14 was identified. In the neurons isolated from mice trigeminal root ganglion, the Na+ current amplitude was reduced by 80% under whole cell patch-clamp recording. There were no apparent modifications to the gating mechanism in the presence of rBmalphaTX14. Although BmalphaTX14 shared a high amino acid sequence similarity with other typical alpha-toxins, it has different effects on neurons. Further electrophysiological analysis suggested that rBmalphaTX14 selectively blocked Na+ channels and is a member of a new group of scorpion toxins.

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“…More recently, several scorpion toxins were expressed in E. coli system as inclusion body [13] and soluble fusion protein with low yield and complicate purification procedures [14]. In this paper we described a simple way to get high yield active neurotoxins through E.Coli expression system by selecting expression vector, modification the genetic codons, prolonging the expression time and the convenient purification procedures combined with affinity chromatography and HPLC.…”

Section: Discussionmentioning

confidence: 99%

High-Level Expression and Purification of an Analgesic Peptide from Buthus martensii Karch

Cao¹,

Wang²,

Xiao³

et al. 2007

PPL

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BmK AngM1, a scorpion peptide isolated from Buthus martensii Karch was reported to exhibit potential analgesic effect. But the relative low content of this toxin in crude venom limits its further characterization. In this study, we constructed an expression vector and transformed into E.coli. The BmK AngM1 was expressed as a fusion protein in the soluble fraction and was purified by Nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. We purified 25 mg recombinant BmK AngM1 (rBmK AngM1) from 1 L bacterial culture. The molecular weight of rBmK AngM1 determined by ESI-MS was 7240.4 Da which was the expected size for correctly processed. Analgesic bioassay studies of rBmK AngM1 exhibited its potential analgesic effect comparable to that of the natural BmK AngM1 peptide.

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Expression of an Insect Excitatory Toxin, BmK IT, from the Scorpion, Buthus martensii Karsch, and its Biological Activity

Cited by 15 publications

References 11 publications

Identification of a Novel Class of Nicotinic Receptor Antagonists

Identification of a Novel Class of Nicotinic Receptor Antagonists

Functional analysis of the α-neurotoxin, BmαTX14, derived from the Chinese scorpion, Buthus martensii Karsch

High-Level Expression and Purification of an Analgesic Peptide from Buthus martensii Karch

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