Gene cloning and sequencing of BmK AS and BmK AS-1, two novel neurotoxins from the scorpion Buthus martensi Karsch.

Based on the known amino acid sequences of BmK AS and BmK AS-1, 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' RACE were cloned and sequenced, and the full length cDNA sequences of BmK AS and BmK AS-1 were then completed by overlapping their two partial cDNA sequences, respectively. The predicted amino acid sequences both consist of 85 amino acid residues including a putative signal peptide of 19 residues and a mature toxin of 66 residues. They are different in 17 amino acid residues, among them 11 residues in the mature toxin. The predicted amino acid sequences of BmK AS and BmK AS-1 were almost consistent with those determined and revised (personal communication), only different in one and two residues at their COO-terminal parts, respectively. Based on the determined cDNA sequences, and using the total DNAs isolated from the scorpion venom glands as a template, the genomic DNAs of BmK AS and BmK AS-1 were also amplified by PCR and sequenced. It showed that no intron was inserted in their open reading frames, while in the exon of signal peptide sequences of other Na+, K+ and Cl- channel toxins from the same scorpion, an intron is usually found. However, the Northern blot hybridization results indicated that the sizes of their mRNA should be around 800 bp. Their extra sequences around 400 bp which might function as an intron should be located at their 5' untranslated regions.     

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 .     

A novel short-chain peptide BmKX from the Chinese scorpion Buthus martensi Karsch, sequencing, gene cloning and structure determination.

Scorpion venom is a rich source of bioactive peptides. From the venom of Chinese scorpion Buthus martensi Karsch (BmK), a novel short chain peptide BmKX of 31-amino acid residues was purified, and its amino acid sequence and gene structure were determined. The gene of BmKX was composed of two exons interrupted by an 86-bp intron at the codon-7 upstream of the mature peptide. Although its gene structure is similar to those of other known scorpion toxins, its amino acid sequence, especially the cysteine framework, is different from those of all other known subfamilies of short-chain scorpion toxins. The solution structure of BmKX, determined with two-dimensional NMR spectroscopy, shows that BmKX also forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins, consisting of a short 3(10)-helix and a two-stranded antiparallel beta-sheet, and the short N-terminal segment forms a pseudo-strand of the beta-sheet. However, the orientation between the helix and the beta-sheet is significantly different from the others, which might be the reason for its unique but still unclear physiological function.     

Purification of two depressant insect neurotoxins and their gene cloning from the scorpion Buthus martensi Karsch

Abstract: Insect‐specific neurotoxins are important components of scorpion venoms. In this study, two toxins from the scorpion Buthus martensi Karsch (BmK) were purified. They shared high sequence homology with other depressant insect toxins and were designated BmK ITa and BmK ITb, respectively. They were able to suppress the action potential of cockroach isolated axon, which is due to a decrease in the peak sodium current. Furthermore, the effect of BmK ITb was lower than that of BmK ITa, and some of the electrophysiological characteristics of BmK ITb even resemble that of excitatory insect toxins. Their primary structures were determined by N‐terminal partial sequence determination and cDNA cloning. The differences in their structures, especially the 31st residues, may result in the unique activity of BmK ITb.     

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.     

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.     

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.     

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.     

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.     

Lack of effect of a neurotoxin from the scorpion Buthus martensi Karsch on nerve fibers of this scorpion

A neurotoxin (BmK I) was purified from the venom of the scorpion species Buthus martensi Karsch. Effects of this toxin on the excitability of the abdominal nerve fibers of the same scorpion were examined. The toxin had no effect at all on the action and resting potentials recorded intracellularly even at a concentration as high as 100 microM. A similar result was obtained through optical measurements of the action potential using a potential sensitive dye. Sea anemone toxin II (8 microM) had no effect on nerve excitability either. However, tetrodotoxin (50 nM) reversibly suppressed the action potential and grayanotoxin II (20 microM) induced a sustained depolarization of the nerve membrane which resulted in a reversible suppression of the action potential. BmK I at a concentration of 0.1 microM greatly prolonged the action potential in the crayfish giant axon. We conclude that the Na channel of nerve fibers of this scorpion is totally insensitive to the neurotoxin in this scorpion's venom.     

东亚钳蝎中新分离的毒素BmTx3B抑制大鼠海马神经元延迟整流性钾电流

目的：研究从东亚钳蝎毒素中新分离的短肽BmTx3B对电压门控性钾通道的作用．方法：在酶解打散的新生大鼠海马细胞,采用全细胞电压箝位方式记录,并根据动力学特性分离二种电压依赖性钾电流．结果：BraTx3B(10-100μmol／L)选择地抑制延迟整流性钾电流(IK),不影响瞬时性快钾电流(IA)．此抑制作用是可逆的,呈现浓度依赖性,但无电压依赖性．BmTx3B对延迟整流性钾电流的稳态激活和稳态失活的动力学特性无影响．结论：蝎毒短肽BmTx3B选择地抑制海马神经元延迟整流性钾通道。     

东亚钳蝎神经毒素基因的序列相似性分析

目的对东亚钳蝎(Buthus martensii Karsch)神经毒素基因进行多序列比对,找到基因的结构特征,在此基础上对序列进行聚类分析,进而推测基因结构与功能的关系。方法搜集目前已知的东亚钳蝎神经毒素基因序列,利用Clustal X1.83软件进行多序列联配,然后用MEGA软件对基因组基因进行聚类分析。结果与结论东亚钳蝎神经毒素基因结构与功能密切相关,而且结构符合断裂基因的基本规律,如果这些基因结构基本一致,是由2个外显子和1个内含子组成,内含子的位置保守,位于信号肽中,基因长度的变化由内含子长度决定。     

A K~ channel-blocking peptide from venom of Chinese scorpion Buthus martensii Karsch

ＡＫ＋ｃｈａｎｎｅｌｂｌｏｃｋｉｎｇｐｅｐｔｉｄｅｆｒｏｍｖｅｎｏｍｏｆＣｈｉｎｅｓｅｓｃｏｒｐｉｏｎＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ１ＷＵＧｏｎｇ，ＷＥＩＤｏｎｇＳｈｅｎｇ２，ＨＥＦａＨｕ，ＨＵＧｕｏＹｕａｎ２，ＷＵＨｏｕＭｉｎｇ...     

马氏钳蝎毒素中的一个钾通道阻断肽

AIM: To purify and characterize a potassium channel blocker (BmP-3) from the venom of Chinese scorpion Buthus martensii Karsch. METHODS: 1. Purification was carried out by gel-filtration, cation-exchange, and reversed-phase chromatographies. N-terminal was directly sequenced by double-coupling manual method. Molecular weight was determined on an electrospray ionization mass spectrometer. Amino acid composition was analyzed after acidic hydrolysis for 20 h in HCl 6 mol.L-1 at 110 degrees C. 2. Toxicity tests were conducted in mice and cockroaches. 3. The inhibitory effects of BmP-3 on K+ channels were tested in acutely dissociated rat hippocampal pyramidal neurons using whole-cell patch-clamp configuration. RESULTS: 1. A pure peptide (BmP-3, 8.1 mg) was obtained, about 0.08% of total proteins of the venom. The N-terminal sequences were VGCEE and the molecular weight was 2938 in ESI-mass spectra. 2. No death occurred at the dosage of 200 micrograms in mice and 8 micrograms in cockroaches. 3. The peptide at 10 mumol.L-1 reduced the peak outward K+ currents by 63% +/- 4% in vitro. CONCLUSION: BmP-3 inhibited K+ channels.     

Purification and pharmacological characterization of BmKK2 (alpha-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch.

BmKK2 (alpha-KTx 14.2) is one of the novel short-chain peptides found in molecular cloning of a venom gland cDNA library from Asian scorpion Buthus martensi Karsch. Based upon its amino acid sequence, the peptide was proposed to adopt a classical alpha/beta-scaffold for alpha-KTxs. In the present study, we purified BmKK2 from the venom of B. martensi Karsch, and investigated its action on voltage-dependent K+ currents in dissociated hippocampal neurons from neonatal rats. BmKK2 (10-100 microM) selectively inhibited the delayed rectifier K+ current, but did not affect the fast transient K+ current. The inhibition of BmKK2 on the delayed rectifier K+ current was reversible and voltage-independent. The peptide did not affect the steady-state activation of the current, but caused a depolarizing shift (about 9 mV) of its steady-state inactivation curve. The results demonstrate that BmKK2 is a novel K+ channel-blocking scorpion peptide.     

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 role of Ser54 in the antinociceptive activity of BmK9, a neurotoxin from the scorpion Buthus martensii Karsch

Residue 54 has been shown to be important for bioactivity in several toxins. However, its role in the antinociceptive activity of toxins has not been evaluated yet. In this study, site-directed mutagenesis and mouse acetic acid writhing test were used to investigate the role of Ser54 in the antinociceptive activity of BmK9 neurotoxin from the Buthus martensii Karsch scorpion. Detailed mutagenesis analysis revealed that substitution of Ser54 by various polar amino acids produced no significant change in the antinociceptive activity, while all substitutions of nonpolar amino acid for Ser54 led to a significant loss of antinociceptive activity. Following the conformational analysis, it was suggested that Ser54 in BmK9 plays a functional role in the antinociceptive activity, the residue exerts its effect by means of a side-chain hydrogen bond.     

Purification,characterization of two peptides from Buthus martensi Karch

Abstract: A new peptide named Martentoxin I and an analogue Martentoxin were purified and characterized from the venom of Buthus martensi Karch. Martentoxin I consisted of 36 amino acid residues with molecular mass as 3908.0 Da determined by matrix‐assisted laser desorption ionization time‐of‐flight‐MS. The amino acid sequence was determined as GLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY by Edman degradation. Martentoxin consisted of 37 amino acid residues with a molecular mass as 4055.3 Da and it showed highly sequence identity to Martentoxin I as FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY. Estimation from circular dichroism spectra indicated Martentoxin I owned 18.0%α‐helix, 53.0%β‐sheet structure and 3.9% turn while Martentoxin contained 13.3%α‐helix, 64.3%β‐sheet structure and 1.1% turn. The toxicity assay showed both peptides had no toxic effects on mice up to the dose of 10 mg/kg. Electrophysiological studies showed that Martentoxin I and Martentoxin at the concentration of 1 μm significantly inhibited voltage‐dependent Na⁺ current (I_Na) and voltage‐dependent delayed rectifier K⁺ current (I_K) but had no effects on transient K⁺ current (I_A). Both interactions with Na⁺ and K⁺ channels were irreversible.     

Precursor nucleotide sequence and genomic organization of BmTXKS1, a new scorpion toxin-like peptide from Buthus martensii Karsch.

Scorpion venom contains a variety of small peptides, which can modulate Na+, K+, Ca2+ and Cl- channel conductance in excitable and non-excitable tissues. A novel full-length cDNA encoding a new toxin-like peptide (named BmTXKS1) was isolated from the venom gland cDNA library of Buthus martensii Karsch. The precursor consists of 60 amino acid residues, with a putative signal peptide of 28 residues and an extra residue, and a mature peptide of 31 residues with an amidated C-terminal. BmTXKS1 shared close homology with BmP01 in 5'UTR and the region encoding the putative signal peptide; especially, the positions of six cysteines are highly conserved among BmTXKS1, PbTX1 and P01-type subfamily of scorpion K+ channel toxins, suggesting that they all should present a common three-dimensional fold, namely the Cysteine-Stabilized alphabeta(CSalphabeta) motif. By PCR amplification of the genomic region encoding BmTXKS1, we have confirmed the identity of our cloned cDNA, and found that BmTXKS1 gene contains an intron, which is completely identical with that of the characterized scorpion K+-channel-ligands in the size, consensus junctions, putative branch point and A+T abundance.