Scorpion Buthus Martensii Karsch Research Articles

Solution structure of BmKαTx11, a toxin from the venom of the Chinese scorpion Buthus martensii Karsch

The solution structure of BmKαTx11 presented by this paper is distinctive from any other structures of wide-type scorpion α-toxins reported so far, for its trans-9,10 peptide bond conformation is accompanied by ‘protruding’ topology of the ‘NC-domain’. The orientation of the C-tail of BmKαTx11 is obviously different from that of classical α-toxins (e.g., AaH2, BmK-M8), despite the fact that they share common trans conformation of peptide bond between residues 9 and 10. Accordingly, there must be other structural factors dominating the orientation of the C-tail except the conformation of peptide bond 9–10. Our study reveals that residues at position 58 play an important role in it, and different type of residues at this position (e.g., Lys, Arg, Met, Ile) result in different spatial relationship between the C-terminus and the ‘five-residue-turn’ and then different topology of the ‘NC-domain’, therefore residues at position 58 are believed to function as structure and bioactivity switch for specificity of scorpion α-toxins. The mechanism for stabilizing the geometry of the ‘NC-domain’ in wide-type scorpion α-toxins is also discussed.

Upregulation of PTEN involved in scorpion venom-induced apoptosis in a lymphoma cell line

We investigated whether the venom of the scorpion Buthus martensii Karsch (BmK) inhibited growth of human lymphoma cells by inducing apoptosis, and studied possible signal pathways involved in this cell death. BmK venom selectively reduced the viability of Raji and Jurkat cells, and had low toxicity to human peripheral blood lymphocytes. Flow cytometry showed that BmK venom-induced apoptosis and G0/G1 cell cycle arrest in Raji and Jurkat cells. In Raji cells, BmK venom upregulated the expression of PTEN accompanied by decreased levels of Akt and Bad phosphorylation. Treatment with BmK venom and LY294002 (an inhibitor of Akt) synergistically enhanced apoptosis. The expression of p27 was increased in both PTEN-positive Raji and PTEN-negative Jurkat cells exposed to BmK venom. The results indicate that key regulators in BmK venom-induced apoptosis are PTEN, acting through downregulation of the PI3K/Akt signal pathway, in Raji cells and p27 in Jurkat cells.

Purification and N-terminal sequence of a serine proteinase-like protein (BMK-CBP) from the venom of the Chinese scorpion ( Buthus martensii Karsch)

A serine proteinase-like protein was isolated from the venom of Chinese red scorpion ( Buthus martensii Karsch) by combination of gel filtration, ion-exchange and reveres-phase chromatography and named BMK-CBP. The apparent molecular weight of BMK-CBP was identified as 33 kDa by SDS-PAGE under non-reducing condition. The sequence of N-terminal 40 amino acids was obtained by Edman degradation. The sequence shows highest similarity to proteinase from insect source. When tested with commonly used substrates of proteinase, no significant hydrolytic activity was observed for BMK-CBP. The purified BMK-CBP was found to bind to the cancer cell line MCF-7 and the cell binding ability was dose-dependent.

Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the α-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC 50 = 0.79 nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure–function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.

Cloning, expression, and pharmacological activity of BmK AS, an active peptide from scorpion Buthus martensii Karsch

BmK AS is a beta long-chain scorpion peptide from the venom of Buthus martensii Karsch (BmK). It was efficiently expressed as a soluble and functional peptide in Escherichia coli, and purified by metal chelating chromatography. About 4.2 mg/l purified recombinant BmK AS could be obtained. The recombinant BmK AS maintained a similar analgesic activity to the natural one in both the mouse-twisting test and hot-plate procedure. It also exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. BmK AS is the first long-chain scorpion peptide reported to have antimicrobial activity, and is a valuable molecular scaffold for pharmacological research.

Purification and characterization of an analgesic peptide from Buthus martensii Karsch

The scorpion Buthus martensii Karsch (BmK) has been one of the indispensable materials in Chinese traditional medicine for thousands of years. In this work, an analgesic peptide was purified through four continuous chromatographic steps. The mouse twisting test was used to identify the target peptides in every separation step. The molecular weight, isoelectric point, and N-terminal residues of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was also cloned by rapid amplification of cDNA ends from the cDNA pool of scorpion glands. This peptide was identical to BmK AS, an agonist of rabbit skeletal muscle ryanodine receptors. Preliminary pharmacodynamics revealed the following: the dose-effect curve plotted by the mouse twisting test showed an ED(50) of 1.42 mg/kg; and the time-effect curves plotted by a hot plate procedure showed a similar effect to the painkiller morphine. We report a purification procedure that yields substantial amounts of natural BmK AS having high activity. BmK AS has the potential to become a new analgesic medicine.

Development of an improved preparation and an enzyme-linked immunosorbent assay for anti-neuroexcitation peptide (ANEP)

Anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. However, the expression of recombinant ANEP in Escherichia coli results in the formation of insoluble aggregates known as inclusion bodies. Here, we describe a novel method for the preparation of ANEP which maximizes the yields of recombinant peptide in a soluble and active form. A non-fusion expression plasmid pNJUTRX-1-ANEP-His 6 encoding recombinant ANEP with a His 6-tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. This effective method solved the problem of a lack of sufficient active peptide which, until now, has hampered the further research and development. In order to develop an immunoassay method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP and purified by protein A affinity chromatography. It was confirmed that the antibody reacted with recombinant ANEP by both Western blotting and ELISA results. Using purified antibody, the immunoassay method was developed.

Therapeutic potential of chlorotoxin-like neurotoxin from the Chinese scorpion for human gliomas

Chlorotoxin, one of the key toxins in scorpion Leiurus quinquestriatus venom, has been shown to bind specifically to glioma cell surface as a specific chloride channel blocker. In this study, a purified, recombinant chlorotoxin-like peptide from the scorpion Buthus martensii Karsch (named rBmK CTa) was characterized by in vivo and in vitro studies. The results from cell proliferation assay with human glioma (SHG-44) cells showed that rBmK CTa inhibits the growth of glioma cells in a dose-dependent manner, with an IC 50 value of approximately 0.28 μM. Under the same conditions, the IC 50 value for normal astrocytes increased to 8 μM. This clearly indicated that rBmK CTa had specific toxicity against glioma cells but not astrocytes. Results from whole-cell patch-clamp recording showed that chloride current in SHG-44 was inhibited by rBmK CTa in a voltage-dependent manner and percent inhibitions for the blocking action of rBmK CTa (0.07 and 0.14 μM) on I Cl was 17.64 ± 3.06% and 55.86 ± 2.83%, respectively. Histological analysis of rBmK CTa treated mice showed that brain, leg muscle and cardiac muscle were the target organs of this toxin. These results suggest that rBmK CTa may have potential therapeutic application in clinical treatment of human glioma. It represents an approach for developing a novel therapeutic agent.

NMR solution structure of BmK-βIT, an excitatory scorpion β-toxin without a ‘hot spot’ at the relevant position

BmK-βIT (previously named as Bm32-VI in the literature), an excitatory scorpion β-toxin, is purified from the venom of the Chinese scorpion Buthus martensii Karsch. It features a primary sequence typical of the excitatory anti-insect toxins: two contiguous Cys residues (Cys37-Cys38) and a shifted location of the fourth disulfide bridges (Cys38-Cys64), and demonstrates bioactivity characteristic of the excitatory β-toxins. However, it is noteworthy that BmK-βIT is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is the first example having a non-glutamate residue at the relevant position in the excitatory scorpion β-toxin subfamily. The 3D structure of BmK-βIT is determined with 2D NMR spectroscopy and molecular modeling. The solution structure of BmK-βIT is closely similar to those of BmK IT-AP and Bj-xtrIT, only distinct from the latter by lack of an α 0-helix. The surface functional patch comparison with those of BmK IT-AP and Bj-xtrIT reveals their striking similarity in the spatial arrangement. These results infer that the functional surface of β-toxins is composed of two binding regions and a functional site. The main binding site is consisted of hydrophobic residues surrounding the α 1-helix and its preceding loop, which is common to all β-type scorpion toxins affecting Na + channels. The second binding site, which determines the specificity of the toxin, locates at the C-terminus for excitatory insect β-toxin, while rests at the β-sheet and its linking loop for anti-mammal toxins. The functional site involved in the voltage sensor-trapping model, which characterizes the function of all β-toxins, is the negatively charged residue Glu15.

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

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.

Characterization of a novel cDNA encoding a short venom peptide derived from venom gland of scorpion Buthus martensii Karsch: Trans-splicing may play an important role in the diversification of scorpion venom peptides

A novel cDNA clone (named BmKT-u) which is a hybrid molecule of the 5′-terminal region of BmKT’ cDNA and the 3′-terminal region of an undocumented cDNA (named BmKu), was isolated from a cDNA library made from the venom gland of scorpion Buthus martensii Karsch. BmKT-u codes for a 30 amino acid residue precursor peptide composed of a 20-residue signal sequence, and a putative 10-residue novel mature peptide. Northern blot hybridization showed BmKT-u cDNA is generated from a transcript. RT-PCR experiments excluded the possibility that BmKT-u cDNA is an artifact generated during reverse transcription. Genomic amplifications performed with three pairs of BmKT-u gene-specific primers showed the BmKT-u gene does not exist in the genome of the scorpion as a single transcriptional unit. Genomic cloning for BmKT’ showed that the BmKT’ gene contains an intron of 509 bp inserted into the region encoding the C-terminal region of the signal peptide. A sequence alignment comparison of the cDNA of BmKT-u with genomic BmKT’ revealed that the junction site of the hybrid molecule is located at the 5′-splicing site of the intron. The data suggest that the BmKT-u transcript is a naturally occurring mature mRNA that is generated by trans-splicing. Trans-splicing may contribute to the diversity of venom peptides from venomous animals.

Genomic Sequence Analysis and Organization of BmKαTx11 and BmKαTx15 from Buthus martensii Karsch: Molecular Evolution of α-toxin genes

Based on the reported cDNA sequences of BmKalphaTxs , the genes encoding toxin BmKalphaTx11 and BmKalphaTx15 were amplified by PCR from the Chinese scorpion Buthus martensii Karsch genomic DNA employing synthetic oligonucleotides. Sequences analysis of nucleotide showed that an intron about 500 bp length interrupts signal peptide coding regions of BmKalphaTx11 and BmKalphaTx15. Using cDNA sequence of BmKalphaTx11 as probe, southern hybridization of BmK genome total DNA was performed. The result indicates that BmKalphaTx11 is multicopy genes or belongs to multiple gene family with high homology genes. The similarity of BmKalpha-toxin gene sequences and southern hybridization revealed the evolution trace of BmKalpha-toxins: BmKalpha-toxin genes evolve from a common progenitor, and the genes diversity is associated with a process of locus duplication and gene divergence.

Adaptive Evolution after Gene Duplication in α-KT × 14 Subfamily from Buthus martensii Karsch

A series of isoforms of alpha-KT x 14 (short chain potassium channel scorpion toxins) were isolated from the venom of Buthus martensii Karsch by RACE and screening cDNA library methods. These isoforms adding BmKK1--3 and BmSKTx1--2 together shared high homology (more than 97%) with each other. The result of genomic sequence analysis showed that a length 79 bp intron is inserted Ala codes between the first and the second base at the 17th amino acid of signal peptide. The introns of these isoforms also share high homology with those of BmKK2 and BmSKT x 1 reported previously. Sequence analysis of many clones of cDNA and genomic DNA showed that a species population or individual polymorphism of alpha-KT x 14 genes took place in scorpion Buthus martensii Karsch and accelerated evolution played an important role in the forming process of alpha-KT x 14 scorpion toxins subfamily. The result of southern hybridization indicated that alpha-KT x 14 toxin genes existed in scorpion chromosome with multicopies. All findings maybe provided an important evidence for an extensive evolutionary process of the scorpion "pharmacological factory": at the early course of evolution, the ancestor toxic gene duplicated into a series of multicopy genes integrated at the different chromosome; at the late course of evolution, subsequent functional divergence of duplicate genes was generated by mutations, deletions and insertion.

Molecular basis of the mammalian potency of the scorpion α‐like toxin, BmK M1

In-depth structure-function studies of voltage-gated Na+ channels and peptide toxins are continuously increasing our understanding of their interaction. In this study, an effective yeast expression system was used to study the role of 14 N- and C-terminal residues from the alpha-like toxin BmK M1 from the Chinese scorpion Buthus martensii Karsch. With the use of site-directed mutagenesis, all of these residues were individually substituted by one or more amino acids, resulting in a total of 19 mutants. These were then subjected to a bioassay on mice, an elaborate electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5, and para), and a circular dichroism analysis. Our results reveal large mutant-dependent differences that emphasize important and specific roles for the studied residues. By mutating single amino acids, we were able to redirect the alpha-like characteristics of BmK M1 (active on both mammals and insects) to either much higher mammal specificity or, in a few cases, total insect specificity. This study therefore represents a thorough mapping and elucidation of three epitopes that underlie the molecular basis of the mammalian and insecticidal potency of the scorpion alpha-like toxin, BmK M1 on voltage-gated Na+ channels.

Structure of an excitatory insect-specific toxin with an analgesic effect on mammals from the scorpionButhus martensiiKarsch

BmK IT-AP is an excitatory insect-specific beta-toxin with analgesic effect from the Chinese scorpion Buthus martensii Karsch (BmK) and consists of 72 residues cross-linked by four disulfide bridges. The crystal structure of BmK IT-AP has been determined at a resolution of 2.6 A by molecular replacement. Compared with the mammal-selective alpha-toxins consisting of 64 residues from the scorpion BmK, the general fold of IT-AP features an additional one-and-a-half turn alpha-helix at the C-terminal residues 59-65 with a shifted disulfide bridge Cys38-Cys64. The extension and 'wiggling' of the C-terminal segment led to a reshaping of the bioactive surface, including the complete destruction of the active site RC comprising the reverse turn (8-12) and C-terminal residues 58-64, the disappearance of an active surface formed by two aromatic residues Trp38 and Tyr42 and the covering of the conserved aromatic cluster Tyr5, Tyr35 and Trp47, which are all critical for the structure and function of mammal-selective alpha-toxins. Bj-xtrIT, the only other excitatory insect-specific toxin whose three-dimensional structure has been determined, is distinct from BmK IT-AP. A functionally important five-residue alpha-helix (alpha0) formed by residues 17-21 in Bj-xtrIT is deleted in BmK IT-AP and helix alpha1 is immediately connected to Cys16 through two residues Leu17 and Phe18. Accordingly, the functional surface of this region in Bj-xtrIT has also been reshaped in IT-AP, which implies subtle differences between BmK IT-AP and Bj-xtrIT in binding to the receptor, although most other critical residues for structure and function adopt almost identical conformations. The crystal structure of IT-AP also forms a sound basis for further study of the structure-function determinants of the analgesic effect.

Expression and purification of the BmK Mm2 neurotoxin from the scorpion Buthus martensii Karsch and its biological activity test

The gene encoding neurotoxin (BmK Mm2) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21 (DE3) at a level of 1.6 mg/L using expression plasmid pExSecI system. SDS–PAGE analysis of the cell lysate confirmed that gene BmK Mm2 was expressed in soluble form and the expressed production was secreted into Luria–Bertani (LB) culture medium from Escherichia coli. According to the characters of pExSecI expression system, the IgG binding domain-ZZ of Protein A is fused to the N-terminal of BmK Mm2. Recombinant BmK Mm2 (ZZ-BmK Mm2, p I 6.81, 22.007 kDa) was purified rapidly and efficiently by IgG–Sepharose 6 Fast Flow and Superdex-75 gel filtration chromatography, produced a single band on SDS–PAGE. Western blot analysis demonstrated that this protein was recombinant BmK Mm2. The results of MTT assay, morphological observation of nucleus and single cell gel electrophoresis showed that the expressed recombinant BmK Mm2 was toxic for glial cells of mice, which indicate that it has biological activity.

Purification, characterization and 1H NMR resonance assignment of an α‐like neurotoxin BmK 16 from the venom of chinese scorpion Buthus martensii karsch

AbstractA natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel‐filtration, ion exchange and reversed phase chromatography. The sequence of the N‐terminal 8 amino acid residues was determined by Edman degradation. Using the TV‐terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N‐terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues′ sequence of the precursor peptide alpha‐neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The sequence‐specific resonance assignment of BmK 16 was achieved and the ′intact sequence of BmK 16 was determined as followings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α‐like scorpion neurotoxin.

Crystallization and preliminary crystallographic study of rBmKalphaIT1, a recombinant alpha-insect toxin from the scorpion Buthus martensii Karsch.

Alpha-insect scorpion toxins are a distinct group of scorpion neurotoxins for which no crystal structures are yet available. A novel alpha-insect toxin named BmKalphaIT1 from the scorpion Buthus martensii Karsch (BmK) has been expressed as an inclusion body in Escherichia coli and purified by chromatography after renaturation. Recombinant BmKalphaIT1 (rBmKaIT1) was crystallized using the vapour-diffusion technique in hanging drops at 296 K. The crystals, which were grown in sodium phosphate, belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 30.24 (1), b = 36.51 (3), c = 57.08 (2) A. Diffraction data were collected to 2.1 A resolution using synchrotron radiation. There appears to be one rBmKalphaIT1 molecule in the asymmetric unit.

Expression, purification and functional characterization of a recombinant scorpion venom peptide BmTXKβ

BmTXKβ, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKβ protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKβ fusion protein released an approximate 6.5 kDa protein which was the expected size for correctly processed. About 2 mg purified recombinant BmTXKβ protein (rBmTXKβ) was produced from 1 l bacterial culture, using this expression and purification system. The function of rBmTXKβ was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKβ inhibited the transient outward current ( I to) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKβ prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.

Expression of an antitumor–analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli

The gene encoding a putative mature antitumor–analgesic peptide (AGAP) from the venom of the Chinese scorpion Buthus martensii Karsch was obtained by polymerase chain reaction (PCR) according to its cDNA sequence and expressed in Escherichia coli. While most of the recombinant AGAP was expressed in the form of insoluble inclusion body. The recombinant AGAP was purified to homogeneity by metal chelating affinity chromatography. Pharmaceutical tests showed that the recombinant AGAP has both analgesic and antitumor activities on mice.