Expression and Purification of the BmK M1 Neurotoxin from the Scorpion Buthus martensii Karsch

Shao, Feng; Xiong, Ye; Zhu, Rong-Huan; Ling, Minhua; Chi, Cheng-Wu; Wang, Dacheng

doi:10.1006/prep.1999.1127

Cited by 32 publications

(33 citation statements)

References 30 publications

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“…Site-directed Mutagenesis of BmK M1-The cDNA of BmK M1 was previously cloned (14) and inserted into pVT102U/␣ (15). According to the sequence of pVT102U/␣-BmK M1, two primers were designed: primer 1 (5Ј-CGTCTAGATAAAAGAAATTCTGTTCGG-3Ј, including a KEX2 protease linker and an XbaI restriction site) and primer 2 (5Ј-CGAAGCTTTTAATGGCATTTTCCTGGTAC-3Ј, with a HindIII site).…”

Section: Methodsmentioning

confidence: 99%

“…BmK M1 has been the subject of different studies: its three-dimensional structure was determined by x-ray crystallography at 1.7-Å resolution (8); the pharmacological properties of Na ϩ channels have recently been investigated (13); and gene cloning and expression of wild-type BmK M1 have also been carried out (14,15).…”

mentioning

confidence: 99%

“…The recombinant plasmid pVT102U/␣-mutant was extracted, sequenced, and transformed into S. cerevisiae S-78 using the LiCl method (18). The expression of the mutants was carried out using a described previously procedure (15). After fermentation, the supernatant of the culture was adjusted to pH 4.2 with acetic acid.…”

mentioning

confidence: 99%

“…Based on an efficient yeast expression system (15), the importance of the above-mentioned aromatic residues in the scorpion toxin BmK M1 was analyzed by site-directed mutagenesis. The results from mutagenesis and expression, characterization, bioassays, and electrophysiological analysis of the mutants are reported here.…”

mentioning

confidence: 99%

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Importance of the Conserved Aromatic Residues in the Scorpion α-Like Toxin BmK M1

Sun

Bosmans

Zhu

et al. 2003

Journal of Biological Chemistry

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About one-third of the amino acid residues conserved in all scorpion long chain Na ؉ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues ( Scorpion neurotoxins targeting voltage-gated sodium channels are single chain polypeptides composed of 60 -70 amino acids cross-linked by four disulfide bridges. They have been divided into two major classes, ␣-and ␤-toxins. Scorpion ␣-toxins, the most extensively studied group, can prolong the action potential by slowing the inactivation of Na ϩ currents with no direct effect on activation (1-3).According to their different pharmacological properties, the ␣-toxins can be further divided into three subgroups, classical ␣-, ␣-like, and insect ␣-toxins (4, 5). The classical ␣-toxins (e.g. AaH II and Lqh II) are highly toxic to mammals, whereas the insect ␣-toxins (e.g. Lqh ␣ insect toxin) are highly toxic to insects. The more recently characterized ␣-like toxins (e.g. Lqh III and BmK M1) act on both mammals and insects, but are unique in their inability to bind to rat synaptosomes despite a high toxicity by intravenous injection. Although three-dimensional structures for the classical ␣-toxins (6, 7), ␣-like toxins (8, 9), and insect ␣-toxins (10) have been elucidated, in-depth structure-function studies of these long chain toxins using sitedirected mutagenesis are still rare, mainly because of folding problems; and the focus has often been on the charged residues in the toxins (11,12). Here, we report the importance of the conserved aromatic residues in ␣-toxins identified by mutagenesis analysis using the ␣-like toxin BmK M1 as template.

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Section: Methodsmentioning

confidence: 99%

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Importance of the Conserved Aromatic Residues in the Scorpion α-Like Toxin BmK M1

Sun

Bosmans

Zhu

et al. 2003

Journal of Biological Chemistry

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“…The interaction of scorpion toxins with their Na v receptor sites has been the focus of extensive research for several decades (reviewed in Rodriguez de la Vega and Possani 2007), yet molecular and structural details about these interactions were limited and have emerged only with the establishment of cloning procedures for the toxins and of an efficient bacterial expression system (LqhaIT anti-insect a-toxin, Gurevitz and Zilberberg 1994;Zilberberg et al 1997; LqhIT2 and Lqh-dprIT 3 depressant b-toxins, Turkov et al 1997;Strugatsky et al 2005; Bj-xtrIT anti-insect excitatory toxin, Froy et al 1999; Lqhb1 and Css4 anti-mammalian b-toxins, Cohen et al 2005; Lqh3 a-like toxin, Karbat et al 2007; Lqq5 and Aah2/Lqh2 anti-mammalian a-toxins, Banerjee et al 2006;Kahn et al 2009) and later in yeast (e.g., BmK M1 a-like toxin: Shao et al 1999). From this point on the study of the toxins did not depend any longer on tedious purifications from crude venoms or chemical modifications that were limited to reactive residues (reviewed in Gurevitz 2012), but rather enabled substitution of any desired single or combination of amino acid residues and massive production of unmodified and mutant toxins for functional and structural analyses.…”

Section: Emerging View Of the Receptor Sites For Scorpion Alpha And Bmentioning

confidence: 99%

Molecular Description of Scorpion Toxin Interaction with Voltage-Gated Sodium Channels

Gurevitz¹,

Gordon²,

Barzilai³

et al. 2013

Toxinology

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Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na v s) at two pharmacologically distinct sites. Alpha toxins bind at receptor site 3 and inhibit channel inactivation, whereas beta toxins bind at receptor site 4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and competition for receptor sites at Na v subtypes. To elucidate the surface of interaction of the two toxin classes with Na v s and clarify the molecular basis of varying toxin preferences, an efficient expression system was established. Mutagenesis accompanied by toxicity, binding, and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography, uncovered the bioactive surfaces of toxin representatives of all pharmacological groups. Exchange of external loops between channels that exhibit marked differences in sensitivity to various toxins accompanied by point mutagenesis highlighted channel determinants that play a role in toxin selectivity. These data were used in further mapping of the brain channel rNa v 1.2a receptor sites for the beta-toxin Css4 (from Centruroides suffusus suffusus) and the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). On the basis of channel mutations that affected Css4 activity, the known structure of the toxin and its bioactive surface, and using the structure of a potassium channel as template, a structural model of Css4 interaction with the gating module of domain II was constructed. This initial model was the first step in the identification of part of receptor site 4. In parallel, a swapping and a mutagenesis approach employing the rNa v 1.2a mammalian and DmNa v 1 insect Na v s and the toxin Lqh2 as a probe were used to search for receptor site 3. The channel mapping along with toxin dissociation assays and double-mutant cycle analyses using toxin and channel mutants identified the gating module of domain IV as the site of interaction with the toxin core domain, thus describing the docking orientation of an alpha toxin at the channel surface.

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First chemical synthesis of a scorpion α‐toxin affecting sodium channels: The Aah I toxin of Androctonus australis hector

M’Barek

Fajloun

Cestèle

et al. 2004

Journal of Peptide Science

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Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.

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Expression and Purification of the BmK M1 Neurotoxin from the Scorpion Buthus martensii Karsch

Cited by 32 publications

References 30 publications

Importance of the Conserved Aromatic Residues in the Scorpion α-Like Toxin BmK M1

Importance of the Conserved Aromatic Residues in the Scorpion α-Like Toxin BmK M1

Molecular Description of Scorpion Toxin Interaction with Voltage-Gated Sodium Channels

First chemical synthesis of a scorpion α‐toxin affecting sodium channels: The Aah I toxin of Androctonus australis hector

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