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.     

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。     

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.     

Molecular cloning and sequence analysis of cDNAs encoding a beta-toxin-like peptide and two MkTx I homologues from scorpion Buthus martensii Karsch.

Three full-length cDNAs, one encoding the precursor of a beta-toxin-like peptide (named BmKBT) and the other two encoding those of (MkTx I) homologues (named MkTx II and MkTx III, respectively), were isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch, by screening with a cDNA fragment generated by PCR. The encoded precursor of BmKBT contained 83 amino acid residues including a signal peptide of 19 residues, a mature peptide of 63 residues and an extra basic residue (Lys) which have to be removed in the processing step. The deduced amino acid sequence of BmKBT showed 52% homology to that of beta-neurotoxin TsVII isolated from scorpion Tityus serrulatus. However, the positions of disulfide bridges have a little variation between the two peptides. The precursors of MkTx II and MkTx III both contained 85 amino acid residues including a signal peptide of 19 residues, a mature peptide of 64 residues and two extra residues (Gly-Arg) which have to be removed in the processing step, too. There was high sequence similarity (90%) between the two peptides. The sequences of mature MkTx II and MkTx III were highly homologous with MkTx I isolated from scorpion Buthus martensii Karsch, both showing 90% identities.     

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.     

Cloning and characterization of a cDNA sequence encoding the precursor of a chlorotoxin-like peptide from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA sequence encoding the precursor of a venom peptide with homology to chlorotoxin (named BmKCT) was isolated from a cDNA library made from the venom glands of the Chinese Scorpion Buthus martensii Karsch. The encoded precursor of BmKCT was 59 amino acid residues long including a signal peptide of 24 residues and a mature toxin of 35 residues with four disulfide bridges. The sequence of BmKCT is similar (68% identities) to that of chlorotoxin isolated from Leiurus quinguestriatus quinquestriatus. BmKCT is the first report of the cDNA sequences encoding four-disulfide-bridged short-chain toxins from Buthus martensuii Karsch so far.     

Site-directed mutagenesis of the toxin from the Chinese scorpion Buthus martensii Karsch (BmKAS): Insight into sites related to analgesic activity

This study utilized the E. coli expression system to investigate the role of amino acid residues in toxin from the Chinese scorpion--Buthus martensii Karsch (BmKAS). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, ten mutants of BmKAS were obtained by PCR. Using site-directed mutagenesis, all of these residues were substituted with different amino acids. This study represents a thorough mapping and elucidation of the epitopes that form the molecular basis of the toxin's analgesic activity. Our results showed large mutant-dependent differences that emphasize the important roles of the studied residues.     

Prokaryotically expressed Buthus martensii Karsch insect depressant toxin has insecticidal effects.

An insect depressant toxin Buthus martensii Karsch insect toxin 4 (BmK IT(4)) cDNA was cloned into the prokaryotic expression vector pSW202 and expressed in HB101 host cells. The authenticity of this in vitro expressed peptide was confirmed by Western blotting, mass spectrometry and N-terminal peptide sequencing. Bioassays using growth media supplemented with BmK IT(4) demonstrated that, at the end of the 5-day experimental period, about 77% of the testing cotton bollworm larvae were killed. Furthermore, the average weight picked up by larvae grown on BmK IT(4) containing media amounts only to 0.7% of that of control group. Our results indicate that BmK IT(4) may be used for biological control of insect damages in place of other traditional insecticides.     

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 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.     

A series of bioactivity-variant neurotoxins from scorpion Buthus martensii Karsch: X-ray crystal structure and functional implications

Three bioactivity-variant neurotoxins, BmK M1, M4, and M8, have been purified from venom of the Chinese scorpion Buthus martensii Karsch. They possess distinct toxic activity on mice in vivo with different electrostatic properties. The relative toxicities of BmK M1, M4, and M8 are 13.3:2.5:1, which interestingly correspond to their respective pI values, ranging from basic to acidic, of 9.01, 7.53, and 5.30, respectively. In addition, the X-ray crystal structure of BmK M8, which is acidic and 1100 times less active than the most potent and basic alpha-toxin AaH II, have been determined at 1.85 A resolution and analyzed in detail. In combination with information from chemical modifications and site-directed mutagenesis, the structural comparisons and sequence alignments suggest a multisite binding mode for toxin-receptor interactions, and three "toxic regions" with relevance to the binding process, including Face A, Face B, and Site C. Face A is featured in the presence of several aromatic residues and may be more essential for the binding; Face B may contribute to the high efficacy of the binding process, on which substitution by acidic residues could weaken the toxic activity; Site C is probably involved in binding site specificity. The main residues involved in these regions will be discussed.     

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 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 33kDa 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.     

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.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

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.     

A new insect neurotoxin AngP1 with analgesic effect from the scorpion Buthus martensii Karsch: purification and characterization

Abstract: An insect toxin named BmK AngP1 was purified from the venom of the scorpion Buthus martensii Karsch (BmK). It also shows an evident analgesic effect on mice, but is interestingly devoid of mammalian toxicity. Bioassay showed that the CPU value of AngP1 was 0.01 μg/body (≈ 30 mg) for the excitatory insect toxicity and 43.0% inhibition efficiency for analgesia at a dose of 5 mg/kg. However, even at the dosage of 10 mg/kg no detectable toxicity on mice could be found. The isoelectric point (pI) value for AngP1 was 4.0, and its molecular mass analyzed by MALDI‐TOF MS was 8141.0. The first 15 N‐terminal residues of AngP1 were determined by Edman degradation and showed high similarity to that of other excitatory scorpion insect toxins. The circular dichroism spectroscopy measured on a JASCO J‐720 system showed that there were 10.4% α‐helix, 46.2% β‐strand and 14.1% turn structure in this peptide. Under two conditions single crystals of AngP1 were obtained.     

The isolation and characterization of a peptide that alters sodium channels from Buthus martensii Karsch.

The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography and found to greatly prolong action potentials at nanomolar concentrations when applied to frog and mouse nerves. The N-terminal primary amino acid sequence of one of the peptides, BMK 16(5), was determined. The first 23 amino acids of BMK 16(5) were found to be: VKDGYIADDRNCPYFCGRNAYYD. The two cysteine residues in the sequence appeared as Edman sequence cycle blanks; however, they were assigned to be cysteines due to sequence similarity to other peptide toxins that bind to sodium channels and identification of the presence of cysteines obtained from single time point amino acid analysis. The MW of BMK 16(5) was determined by a Perkin Elmer API 300 LC/MS/MS to be 3,695. The amino acid residues of BMK 16(5) show strong similarity with the first 23 amino acid residues of a number of scorpion alpha neurotoxins. Unlike these neurotoxins, BMK 16(5) possesses a proline residue at position 13 which will likely make it fold in a unique way so as to bind to and alter sodium channels.     

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.