Adaptive evolution after gene duplication

One of the two ribonuclease genes in a leaf-eating monkey has adapted to a role in the digestion of bacterial RNA. Following duplication of the ancestral ribonuclease gene, adaptation occurred through a series of changes in the amino acid sequence of the protein it encodes. This example is a good illustration of how specialization of protein function after gene duplication can be as source of novel protein functions.     

Scorpion toxins from Buthus martensii Karsch all possess a predicted alpha-tight-turn

We have purified a new toxin (BmK 17[4]) from Asian scorpion (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8-12) that is similarly found in two other previously described alpha-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration, ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K+ and Ca2+ channels were blocked, BMK 17[4] acts to selectively alter Na+ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC --. The molar mass of BmK 17[4] was determined by LC/MS/MS to be 7097 Daltons. The N- terminal motif (KPENC), which introduces a reverse turn in residues 8-12, does not appear in previously characterized BmK alpha-toxins and may be characteristic of alpha-like toxins. Sequence similarity database searches were used to test whether the N-terminal sequences of alpha-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (alpha-turn) that is conserved. Sequence similarities with putative polypeptides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon 38, 1653-1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their alpha-turn. A neighbor-joining phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the KPENC motif and the other possesses a modifed version (KPHNC) of it.     

Precursors of three unique cysteine-rich peptides from the scorpion Buthus martensii Karsch

Scorpion venoms contain a large number of small peptides with diverse primary structures and unique pharmacological functions. From a cDNA library prepared from venom glands of the Chinese scorpion Buthus martensii Karsch, clones encoding precursors of three unique cysteine-rich peptides named BmTXKS3, BmTXLP2 and BmAP1 have been isolated and sequenced. These precursors are composed of 54, 94 and 89 amino acids, respectively, containing a signal peptide in their N-termini. Sequence analysis shows that BmTXKS3 and BmTXLP2 are two novel members of a scorpion toxin family sharing cysteine-stabilized α-helical folds. BmAP1 possesses a distinctive cystine framework, which is similar to some serine protease inhibitors and the segments of several extracellular proteins.     

两个东亚钳蝎抗哺乳动物神经毒素的cDNA序列

从我国山东东亚马氏钳蝎尾腺中分离纯化ｍＲＮＡ,经逆转录构建了ＢｍＫ蝎毒ｃＤＮＡ文库。利用ＰＣＲ扩增,筛选到两个抗哺乳动物毒素的ｃＤＮＡ基因,并测定了序列。这两个ｃＤＮＡ阅读框均为２５２ｂｐ组成,可翻译８４肽的毒素前体,包括Ｎ端１９个氨基酸组成的信号肽,６４个氨基酸残芭的成熟毒蛋白,以及Ｃ端一个额外的碱性氨基酸Ａｒｇ。其中由一ＣＤＮＡ所推导的氨基酸序列（ＢｍＫＭ１）一已知的天然毒素ＢｍＫ１蛋白序列完     

东亚钳蝎蝎毒素BmKBT基因组序列的克隆及其分析

东亚钳蝎 (ButhusmartensiiKarsch ,BmK)蝎毒素BmKBT(又名BmKabT)是一个在初级结构上相似于β类哺乳动物毒素和功能接近于α类哺乳动物毒素的Na+ 通道毒素 .基于从毒腺cDNA文库中筛选得到的全长BmKBT前体核苷酸序列设计引物 ,以蝎基因组总DNA为模板进行聚合酶链式反应 (PCR) ,将PCR产物克隆至T载体、测序 .序列分析表明 :在BmKBT信号肽编码区的 3′端的- 4位Gly密码子的第 1位与第 2位碱基中有 1个长 2 2 5nt的内含子 ,插入位点距离该基因的起始密码子 4 6nt ,AT含量为 78 7% ,其内含子可能的剪接分枝位点距离 3′剪接受体位点 4 7nt.内含子的大小及其基因组织结构分析表明 :BmKBT具有与α类哺乳动物毒素类似的基因组织结构 ,进一步说明BmKBT是一个介于α类和β类Na+ 通道毒素之间的中间型蝎毒素 ,可以作为研究蝎毒素分子进化的合适材料     

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.     

A depressant insect toxin with a novel analgesic effect from scorpion Buthus martensii Karsch

A new peptide named BmK dITAP3 from scorpion Buthus martensii Karsch (BmK) has been identified to possess a dual bioactivity, a depressant neurotoxicity on insects and an analgesic effect on mice. The bioassays also showed that the peptide was definitely devoid of the neurotoxicity on mammals, which indicated that the analgesic effect of BmK dITAP3 could not be ascribed to the syndromic effects of a mammalian neurotoxicity. BmK dITAP3 exhibited 43.0% inhibition efficiency of the analgesic effect on mice at a dose of 5 mg/kg and the FPU value of 0.5 microg/body (approximately 30 mg) on the fly larvae. The pI value and the molecular mass determined by MALDI-TOF MS for dITAP3 were 6.5 and 6722.7, respectively. Its first 15 N-terminal residues were determined by Edman degradation, based on which the full amino acid sequence was deduced from the cDNA sequence encoding the peptide with 3'-RACE. Circular dichroism and sequence based prediction analyses showed dITAP3 may have a similar molecular scaffold as the most scorpion toxins but with features of the more beta structures and much less of alpha helix. The details of the purification, characterization and sequencing as well as the sequence comparison with other depressant insect toxins and the correlation between the analgesic effect and the insect toxicity will be reported and discussed, respectively.     

Functional study of active residues scorpion insect toxin BmK IT from Buthus martensii Karsch

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating. An excitatory insect toxin, BmK IT, is not conserved with a glutamate residue at the preceding position of the third Cys residue, and is a toxin with a non-glutamate residue at the relevant position in the excitatory scorpion β-toxin subfamily. In this study, the mutants of recombinant BmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT C-terminal (TKSYCDVQIN) truncated) were achieved by site-directed mutagenesis. Biological activity of BmK IT and its mutants confirmed these residues or peptides played key roles in BmK IT. BmK IT (I25E) could increase the sensitivity of BmK IT, but BmK IT(E15G) could decrease the sensitivity of BmK IT on Sf9 cells. BmK IT truncated C-terminal hydrophobic amino acids could cross the species boundaries and was effective on mammalian C6 cells. To date, several excitatory insect toxins have been isolated and identified from the venom of Buthus martensii Karsch. However, no functional data are available and therefore its classification in the family of excitatory insect toxins remains putative and is just based on its high similarity with the other toxins of this family. These results verified I25, E15 and C-terminal (TKSYCDVQIN) in BmK IT played key roles in the interaction of the BmK IT and its receptor- sodium channels on the surface of insect cells and laid a foundation for further structural and functional analysis of BmK IT.     

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.     

Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500 mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED₅₀ of 0.5 mg/kg.     

Genomic sequence analysis and organization of BmKalphaTx11 and BmKalphaTx15 from Buthus martensii Karsch: molecular evolution of alpha-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.     

Expression, renaturation and functional analysis of an excitatory insect-specific toxin from scorpion Buthus martensii Karsch

The cDNA of BmK IT-AP, an excitatory insect toxin from the scorpion Buthus martensi Karsch that has an analgesic effect on mammalian cells, was expressed in E. coli in the form of an inclusion body. Following denaturation and reduction, the recombinant protein was renatured and purified by liquid chromatography. The authenticity of the recombinant product was confirmed by bioassay and its electrophysiological effect on insect sodium channel.     

Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch

The scorpion venom peptides with no disulfide bridge are rarely identified and poorly characterized so far. Here, we report the identification and characterization of four novel disulfide-bridge-free venom peptides (BmKa1, BmKa2, BmKb1 and BmKn2) from Buthus martensii Kasch. BmKa1 and BmKa2 are very acidic and hydrophilic, showing no any similarity to other proteins, whereas BmKb1 and BmKn2 both are basic, alpha-helical peptide with an amidated C-terminus, showing a little homology with other peptides. Functional tests with synthetic peptide showed that BmKn2 has strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, whereas BmKb1 has weak activity in inhibiting the growth of these bacteria.     

Purification and N-terminal partial sequence of anti-epilepsy peptide from venom of the scorpion Buthus martensii Karsch.

An anti-epilepsy peptide (AEP) was isolated and purified from venom of the scorpion Buthus martensii Karsch. The purification procedure included CM-Sephadex C-50 chromatography, gel filtration on Sephadex G-50 and DEAE-Sephadex A-50 chromatography. Its homogeneity was demonstrated by pH 4.3 polyacrylamide-disc-gel electrophoresis, focusing electrophoresis and SDS/polyacrylamide-disc-gel electrophoresis. The Mr of this peptide, calculated from measurements in SDS/15%-polyacrylamide-disc-gel and SDS/20%-polyacrylamide-disc-gel electrophoresis, is 8300. The isoelectric point is 8.52 by pH 8-9.5-range isoelectric focusing. No haemorrhagic or toxic activities were found. No toxicity was found even after the dose reached 28 mg/kg. The pharmacological tests showed that the AEP had no effect on heart rate, blood pressure or electrocardiogram, but strongly inhibited epilepsy induced by coriaria lactone and cephaloridine. The fluorescence spectrum showed that the peptide has a strong emission peak at 337 nm. Amino acid analysis suggested that the AEP is composed of 66 residues from 18 amino acids and has an Mr of 8290. The sequence of the first 50 N-terminal residues is as follows: Asp-Gly-Tyr-Ile-Arg-Gly-Ser-Asp-Asn-Cys-Lys-Val-Ser-Cys-Leu-Leu-Gly-Asn- Glu-Gly - Cys-Asn-Lys-Glu-Cys-Arg-Ala-Tyr-Gly-Ala-Ser-Tyr-Gly-Tyr-Cys-Trp-Thr-Val- Lys-Leu - Ala-Gln-Asp-Cys-Glu-Gly-Leu-Pro-Asp-Thr-.     

Expression of anti-neuroexcitation peptide III of scorpion Buthus martensii Karsch BmK ANEP III in plants

Anti-neuroexcitation peptide III of Buthus martensii Karsch (BmK ANEP III) has better anti-epileptic and anticonvulsive effects in the test animal models. The present study is aimed at developing transgenic tomato and tobacco lines overproducing the ANEP III protein. Using the molecular cloning technique, the plant expression vector pBI-ANEP III was constructed successfully. The ANEP III expression cassette included a double CaMV 35S promoter with omega enhancers, the ANEP III gene with the Kozak sequence, the ER retention signal and the NOS terminator. Recombinant plasmids were transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw transformation methods. By the Agrobacterium-mediated leaf disc transformation method, tobacco (Nicotiana tabacum) and tomato (Lycopersicum esculentum) lines were transformed. Transformants were screened and confirmed by PCR, RT-PCR and western blotting analysis. It was demonstrated that the ANEP III gene was successfully expressed in the genomic DNA of transgenic plants. The ANEP III protein was detected by immunofluorescence analysis, and the results confirmed the high amount of ANEP III protein, being 0.81 and 1.08% of total soluble proteins in transgenic tobacco and tomato. The study of plants with high expression levels of ANEP III has an important theoretical and practical significance and provides valuable information for establishing a new, economical and effective system for industrial protein production.     

Expression of anti-neuroexcitation peptide (ANEP) of scorpion Buthus martensii Karsch in Escherichia coli

According to the cDNA sequence of anti-neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method, then was cloned into expression plasmid pET28a, fused with His tag at its 3' end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins, while most recombinant ANEP products existed in the form of insoluble inclusion bodies. Coexpression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and Superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti-neuroexcitatory activity.     

Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series.It is medially toxic and belongs to group III α-toxins.The purified sample was crystallized in rhombic space group P61.Using an X-ray diffraction technique,the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution.The model was refined.The final crystallographic R factor was 0.142 and the free R factor was 0.173.The root mean square deviation is 0.001 5 nm for the bond length and 1.753°for the bond angles.64 water molecules were added to the asymmetric unit.The refined structure showed an unusual non-prolyl cis peptide bond at residue 10.The structure was compared with group II α-toxin BmK M8 (an acidic,weak toxin).The potential structural implications of the cis peptide bond were discussed.     

Separation and Purification of Antioxidant Peptides from Enzymatically Prepared Scorpion (Buthus martensii Karsch) Protein Hydrolysates

International Journal of Peptide Research and Therapeutics - The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein...     

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 alpha-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)=0.79nM), 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.     

一种东亚钳蝎碱性神经毒素的纯化和初步晶体学研究

经ＳｅｐｈａｄｅｘＧ－５０和ＳＰ－ＳｅｐｈａｄｅｘＣ－２５两次柱层析,从河南淅川马氏钳蝎毒素中分得一种碱性神经毒素ＢｍＫＭＩ８．等电聚焦和ＳＤＳ电泳显示单一组分,其ｐＩ为９．１,Ｍｒ为７１００．毒性测试结果表明,该组分对小白鼠有较强的毒性,对昆虫也有一定的毒性,已获得该毒素的两种晶型的大单晶,并测定其空间群为Ｐ２＿１２＿１２＿１,晶胞参数为：ＢｍＫＭＩ８－Ａ：ａ＝３６．７,ｂ＝２６．６,ｃ＝５２．０,ＢｍＫＭＩ８－Ｂ：ａ＝３６．６４Ａ,ｂ＝３７．３１,ｃ＝３７．９５,已收集了ＢｍＫＭＩ８－Ｂ分辨率为１．７４的Ｘ射线单晶衍射数据。