Covalent structures of BmK AS and BmK AS-1, two novel bioactive polypeptides purified from Chinese scorpion Buthus martensi Karsch.

Complete amino acid sequences of two novel bioactive polypeptides, each containing 66 amino acid residues, BmK AS and BmK AS-1 purified from the venom of Chinese scorpion Buthus martensi Karsch, have been determined by Edman sequencing and mass spectrometry on native proteins, reduced and S-carboxymethylated proteins and their peptides obtained after cleavage with proteolytic enzymes. Sequence analysis showed 86.4% structural identity between BmK AS and BmK AS-1 and also a high sequence similarity between BmK ASs and AaH IT4, a unique anti-insect toxin and a ligand of Na+ channels obtained from Sahara scorpion A. australis Hector, but poor sequence homology between BmK ASs and those of the known alpha-, beta-type and long-chain insect-selective type scorpion neurotoxins. The positions of four disulfide bridges in BmK AS-1 were established as Cys-12 and Cys-62, Cys-16 and Cys-37, Cys-23 and Cys-44, and Cys-27 and Cys-46, which are the same as those in alpha- and beta-scorpion neurotoxins. These results suggest that BmK ASs and AaH IT4 may form a new group sharing similar structural and functional properties in the family of scorpion neurotoxic polypeptides.     

Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, the gene specific primers were designed and synthesized for 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fragments obtained by 3' and 5' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.     

The gene cloning and sequencing of Bm-12, a chlorotoxin-like peptide from the scorpion Buthus martensi Karsch.

According to the known amino acid sequence of Bm-12, a short chain insect neurotoxin from the venom of the scorpion Buthus martensi Karsch (BmK) with considerable primary sequence homology to chlorotoxin, the gene specific primers were designed and synthesized for 3' and 5'RACE (Rapid Amplification of cDNA Ends). The two partial cDNA fragments obtained by 3' and 5'RACE were cloned and sequenced, and the full length cDNA sequence of Bm-12 was then completed by overlapping these two partial cDNA sequences. The predicted amino acid sequence consists of 59 amino acid residues including a putative signal peptide of 24 residues and a mature toxin of 35 residues. The predicted amino acid sequence of Bm-12 was almost consistent with the determined, different only in one residue at position 27, Lys was replaced by Gly. Based on the determined cDNA sequence, and using the total DNA isolated from the scorpion venom glands as a template, the genomic DNA of Bm-12 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 93 bp present within the signal peptide region.     

The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch.

The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins.     

Cloning and characterization of the cDNA sequences of two venom peptides from Chinese scorpion Buthus martensii Karsch (BmK).

From a cDNA library made from venom glands of Chinese scorpions of Buthus martensii Karsch, full-length cDNAs encoding precursors of two venom peptides have been isolated using a cDNA probe synthesized by polymerase chain reaction. Sequence analysis of the cDNAs revealed that one encoded precursor was 85 amino acid residues long including a signal peptide of 19 residues and a mature peptide (named BmK T) of 66 residues, and another encoded precursor was 84 residues long containing the same length signal peptide and a mature peptide (BmK M4 isoform, named BmK M4') of 64 residues. The analysis of amino acid sequence similarity indicated that the BmK T was homologous with both mammalian and insect toxins from BmK scorpion or other scorpions, and the BmK M4' was highly homologous with the members of the mammalian neurotoxin family of BmK, having two point mutations in amino acid residue sequence compared to BmK M4, a natural toxin from BmK.     

Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.     

Characterization of four distinct monoclonal antibodies specific to BmK AS-1, a novel scorpion bioactive polypeptide.

Four monoclonal antibodies designed as 2#, 3#, 4# and 5# have been raised against a novel bioactive polypeptide BmK AS-1 purified from the Chinese scorpion Buthus martensi Karsch. All of these antibodies exhibited specific affinity with antigen by ELISA and Biosensor assay. Western blot analysis showed that 3# and 4# were able to recognize the denatured antigen, but not 2# and 5#. These antibodies could cross-react with BmK AS, but not with other types of BmK neurotoxins such as BmK I (an alpha-like toxin) and BmK IT (an excitatory insect-selective toxin), and in which only 5# can partially react with BmK IT2 (a depressant insect-selective toxin). Immunocytochemical staining demonstrated that 3#, 4# and 5# antibodies can visualize the antigen bound to the membrane of SK-N-SH neuroblast cells, with the exception of 2#. This suggests that either conformation alteration of receptor binding might be prone to nonvisualization or the epitope recognized by antibody 2# might be overlapped with receptor binding sites of antigen. The antibodies developed in the study should provide powerful new tools for investigating the structure/function relationship and pharmacological mechanism of scorpion neurotoxins.     

蝎毒活性肽BmK AS的基因克隆与表达

目的克隆东亚钳蝎活性肽BmKAS的基因,在大肠杆菌中进行表达。方法利用PCR技术从蝎尾cDNA pool中扩增BmKAS基因,通过基因工程方法构建表达载体pET28a-BmKAS,在E.coli中进行表达,表达产物为包含体。采用分步稀释复性法对表达产物进行体外变复性。结果BmKAS以包含体的形式得到表达,表达量的质量约占菌体总蛋白质量的31%。表达产物复性后,通过离子交换柱层析进一步纯化,达到了电泳纯,收率为1.6 mg.L-1。结论首次对具有多种药理活性的蝎毒活性肽BmKAS进行了表达,并对表达产物进行体外变复性研究,获得了毫克级的收率,满足了深入研究需要。     

cDNA sequence analysis of seven peptide toxins from the spider Selenocosmia huwena.

Seven cDNAs encoding six toxins HWTX-I, HWTX-II, HWTX-IIIa, HWTX-IV, HWTX-V, HWTX-VII and one lectin SHL-I, from the spider Selenocosmia huwena, were cloned and sequenced. On the basis of their amino acid sequences, we designed and synthesized 3' RACE and 5' RACE primer. By overlapping the two partial cDNA sequences obtained by 3' and 5' RACE, their full-length cDNA sequences were obtained. All of the cDNAs of these seven peptides encode a precursor including a potential signal peptide of 21-24 residues, a mature toxin of about 30 residues and an intervening pro region. The prepro regions of HWTX-I, HWTX-IIIa, HWTX-IV, HWTX-V and SHL-I were demonstrated, by the comparison of the cDNA sequences, to have high similarity, which is concert with the similar inhibitor cystine knot motif of HWTX-I, HWTX-IV and SHL-I although their functions are different. It was also demonstrated that, HWTX-II and HWTX-VII share the highly similar prepro region which is different from that of HWTX-I, HWTX-IV and SHL-I. The three dimensional structure of HWTX-II has been determined to exhibit a different motif. This indicates that the seven peptides from S. huwena could be classified into two different superfamilies according to the prepro region of cDNA sequences.     

Cloning and characterization of cDNA sequences encoding two novel alpha-like-toxin precursors from the Chinese scorpion Buthus martensii Karsch.

According to a relative conserved fragment of alpha-scorpion toxins, a degenerate primer was designed and synthesized. Two full-length cDNAs encoding the precursors of two novel putative alpha-like-toxins were then amplified from the total cDNAs of venomous glands of the Chinese scorpion Buthusmartensi Karsch using 3' and 5' RACE (rapid amplification of cDNA ends). The precursors were both composed of 85 amino acid residues, including a putative signal peptide of 19 residues and a mature toxin of 66 residues, respectively. The predicted amino acid sequences of these two toxins show a homology of 82% with each other, and of 55-70% with other BmK-originated alpha-like-toxins. Interestingly, it is rarely seen in other alpha or alpha-like-toxins that: (1) Met residue but not a basic amino acid residue (Arg or Lys) is located on position 58 for BmKalpha2; (2) both toxins are ended with double Gly in the C-terminus.     

Molecular cloning and genomic organization of a K(+) channel toxin from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA encoding the precursor of a K(+) channel toxin (BmTX2) was first isolated from a venom-gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor is composed of a signal peptide of 21 residues and a mature toxin of 37 residues with three disulfide bridges. The genomic gene of BmTX2 was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 81 bp inserted in the region encoding signal peptide.     

东亚钳蝎毒素多肽BmK AS-1对大鼠皮肤痛觉的影响

观察东亚钳蝎 ( Bm K)毒素纯化组分 Bm K F-1 - 3,Bm K F- 1 - 3- 2和 Bm K AS- 1 ( Bm K F- 1 - 3- 2 - 1 )中枢和外周给药对大鼠皮肤痛觉的影响 .方法采用局部皮肤感受野给药 ,以强电流刺激大鼠后肢趾部诱发半膜半腱肌发放 C反应 ,观察对外周神经系统的镇痛作用 ;经脊髓蛛网膜下腔给药 ,以大鼠足跖辐射热痛阈的变化为中枢镇痛效应的观察指标 .实验结果显示 Bm K F- 1 - 3,Bm K F- 1 - 3- 2和 Bm KAS- 1抑制 50 % C反应的剂量为 40 ,2 8.3和 1 0μg,且抑制作用不能被纳洛酮翻转 ;Bm K F- 1 - 3ith无明显提高大鼠足跖辐射热痛阈的作用 ,而 Bm KAS- 1 ith则可显著提高大鼠足跖辐射热痛阈 ,其提高 1 50 %痛阈的剂量约为 1 .2μg,纳洛酮同样对Bm K AS- 1的中枢镇痛效应无翻转作用 .结果提示东亚钳蝎毒素纯化组分 Bm K AS- 1可提高大鼠皮肤痛阈 ,其作用机理有别于阿片肽类物质 .     

The binding of BmK abT,a unique neurotoxin,to mammal brain and insect Na(+) channels using biosensor

The binding properties of BmK abT (a novel neurotoxic polypeptide abT from Chinese scorpion Buthus martensi Karsch), a unique neurotoxin from Chinese scorpion, on mammal brain and insect sodium channels were investigated using the BIAcore assay. Results showed that BmK abT could bind to rat brain synaptosomes with an association rate constant of about 2.49 x 10(6) M(-1) s(-1) and a dissociation rate constant of about 1.57 x 10(-4) s(-1), and to Heliothis nerve cord synaptosomes with an association rate constant of about 1.21 x 10(7) M(-1) s(-1) and a dissociation rate constant of about 0.99 x 10(-3) s(-1). The binding of BmK abT to rat brain synaptosomes could be partially inhibited by increasing the membrane potential, but not by BmK AS (a novel active polypeptide AS from B. martensi Karsch), BmK IT2 (a depressant insect-selective toxin IT2 from B. martensi Karsch), and BmK I (an alpha-like anti-mammal toxin I from B. martensi Karsch). Binding was not modulated by veratridine. In addition, the binding of BmK abT to Heliothis nerve cord synaptosomes was significantly enhanced by increasing the membrane potential and veratridine concentration and was inhibited by BmK IT2, but not by BmK AS or BmK I. The results suggest that BmK abT binds to a distinct receptor site on mammal brain Na(+) channels and associates with a related site for depressant insect-selective toxins on insect sodium channels.     

Nucleotide sequence and structure analysis of a cDNA encoding an alpha insect toxin from the scorpion Leiurus quinquestriatus hebraeus.

A approximately 370 base pair cDNA encoding the alpha insect toxin Lqh alpha IT of the scorpion Leiurus quinquestriatus hebraeus was cloned and sequenced. The deduced amino acid sequence for the putative mature polypeptide is identical to the protein sequence determined chemically (Eitan et al., Biochemistry 29, 5941, 1990). A 19 amino acid signal peptide precedes the 64 amino acid long toxin. Two additional amino acid residues that do not correspond to the purified toxin are found at the COOH-terminus and may imply post-translational modification. The signal peptide region in the present clone differs obviously from that encoding the depressant insect toxin LqhIT2 derived from the same venom, but strongly resembles the leader peptide sequence of an alpha-mammal toxin from the scorpion Androctonus australis.     

BmK86-P1, a New Degradation Peptide with Desirable Thermostability and Kv1.2 Channel-Specific Activity from Traditional Chinese Scorpion Medicinal Material

Thermally processed Buthus martensii Karsch scorpions are a traditional Chinese medical material for treating various diseases. However, their pharmacological foundation remains unclear. Here, a new degraded peptide of scorpion toxin was identified in Chinese scorpion medicinal material by proteomics. It was named BmK86-P1 and has six conserved cysteine residues. Homology modeling and circular dichroism spectra experiments revealed that BmK86-P1 not only contained representative disulfide bond-stabilized α-helical and β-sheet motifs but also showed remarkable stability at test temperatures from 20–95 °C. Electrophysiology experiments indicated that BmK86-P1 was a highly potent and selective inhibitor of the hKv1.2 channel with IC₅₀ values of 28.5 ± 6.3 nM. Structural and functional dissection revealed that two residues of BmK86-P1 (i.e., Lys¹⁹ and Ile²¹) were the key residues that interacted with the hKv1.2 channel. In addition, channel chimeras and mutagenesis experiments revealed that three amino acids (i.e., Gln³⁵⁷, Val³⁸¹ and Thr³⁸³) of the hKv1.2 channel were responsible for BmK86-P1 selectivity. This research uncovered a new bioactive peptide from traditional Chinese scorpion medicinal material that has desirable thermostability and Kv1.2 channel-specific activity, which strongly suggests that thermally processed scorpions are novel peptide resources for new drug discovery for the Kv1.2 channel-related ataxia and epilepsy diseases.     

Nine novel precursors of Buthus martensii scorpion alpha-toxin homologues.

The cDNAs encoding nine novel alpha-toxin homologues were isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). They are rich in AAAA and TTTT elements at the 5' UTRs. The flanking region of the translation initiation codon ATG is AAAATGAA, which is highly conserved in scorpion Na(+), K(+) and Cl(-) channel toxin genes. These putative scorpion alpha-toxins shared 45.5-98.4% homology with the characterized BmK alpha-toxins, and were completely conserved in the positions of all eight cysteines. This showed, together with higher homology at nucleotide level than that at amino acid level, that these toxins may originate from a common ancestor. The discovery of a series of homologues of scorpion alpha-toxin with a different degree of natural mutation in the primary structure will provide us with a valuable system for studying the structure-function relationship of scorpion toxins.     

U-shaped dose-dependent effects of BmK AS,a unique scorpion polypeptide toxin,on voltage-gated sodium channels

Background and purpose:

Buthus martensi Karsch (BmK) AS is a scorpion polypeptide toxin, said to target the voltage-gated sodium channels (VGSCs). However, the mechanism of action of BmK AS on the VGSCs has yet to be defined.

Experimental approach:

We examined the electrophysiological effects of BmK AS in a wide dose range on the rat brain-type VGSC α-subunit, rNav1.2a, heterologously expressed in Xenopus oocytes and on the VGSCs endogenously expressed in the dorsal root ganglion neuroblastoma ND7-23 cell line.

Key results:

In the oocytes, BmK AS depolarized the voltage dependence of activation and inactivation of rNav1.2a at 0.1 and 500 nM whereas these parameters were hyperpolarized at 1 nM. In ND7-23 cells, BmK AS hyperpolarized the voltage dependence of activation and inactivation at 0.1, 1 and 100 nM but not 10 nM. BmK AS also hyperpolarized the voltage dependence of recovery from inactivation at 0.1 and 100 nM and slowed the recovery kinetics at all concentrations, but the effects of 1 and 10 nM were relatively smaller than those at 0.1 and 100 nM. Moreover, the inactivation of VGSCs was potentiated by 10 nM BmK AS in both systems, whereas it was inhibited by 0.1 or 100 nM BmK AS in the oocytes or ND7-23 cells respectively.

Conclusions and implications:

BmK AS modulated the VGSCs in a unique U-shaped dose-dependent manner, which could be due to the opposing effects of binding to two distinct receptor sites on the VGSCs.     

cDNA cloning of two A-superfamily conotoxins from Conus striatus.

The full-length cDNAs of two A-superfamily conotoxins, kappaA-SIVA and alpha-SII, were respectively cloned and sequenced from Conus striatus using 3' RACE and 5' RACE. The cDNA of kappaA-SIVA encodes a precursor of 68 residues, including a signal peptide of 21 residues, a pro-peptide of 17 residues, and a mature peptide of 30 residues with an additional residue Gly which is prerequisite for the amidation of the preceding C-terminal Cys. The cDNA-deduced sequence of alpha-SII is composed of a signal peptide of 21 residues, a pro-peptide of 29 residues, a mature peptide of 19 residues and three additional residues Arg-Thr-Ile at the C-terminus. This tripeptide might be cleaved off by proteolytic processing. Although these two conotoxins belong to different families and target voltage-gated potassium channel and nicotinic acetylcholine receptor, respectively, they share the same signal sequence, and both are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The length of 3' untranslational region of alpha-conotoxin SII was extraordinarily large about 10 times longer than that of kappaA-SIVA with 770 and 75 bp, respectively. The elucidated cDNAs of these two toxins will facilitate a better understanding of the process of their post-translational modifications.     

Characterisation of the genes encoding Aa1 isoforms from the scorpion Androctonus australis.

Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K(+) currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron-exon organisation of the gene corresponding to the AaTX1 cDNAs was also depicted.