[Show abstract][Hide abstract] ABSTRACT: Representing a basal branch of arachnids, scorpions are known as 'living fossils' that maintain an ancient anatomy and are adapted to have survived extreme climate changes. Here we report the genome sequence of Mesobuthus martensii, containing 32,016 protein-coding genes, the most among sequenced arthropods. Although M. martensii appears to evolve conservatively, it has a greater gene family turnover than the insects that have undergone diverse morphological and physiological changes, suggesting the decoupling of the molecular and morphological evolution in scorpions. Underlying the long-term adaptation of scorpions is the expansion of the gene families enriched in basic metabolic pathways, signalling pathways, neurotoxins and cytochrome P450, and the different dynamics of expansion between the shared and the scorpion lineage-specific gene families. Genomic and transcriptomic analyses further illustrate the important genetic features associated with prey, nocturnal behaviour, feeding and detoxification. The M. martensii genome reveals a unique adaptation model of arthropods, offering new insights into the genetic bases of the living fossils.
Full-text · Article · Oct 2013 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: We present an identification key to the scorpion species of Yunnan (China) with notes on the distribution and ecology. Euscorpiops kubani is recorded for the first time for China. The redescriptions of Euscorpiops shidian and Euscorpiops kubani are provided. The number of known scorpion species from Yunnan is raised to nine.
[Show abstract][Hide abstract] ABSTRACT: Malignant gliomas are the most common primary brain tumors associated with significant morbidity and mortality. How to target the tumor in situ, and inhibit tumor cell proliferation and invasion is the key for therapy. Gliomas express a glioma-specific chloride ion channel that is sensitive to toxins including BmKCT. In the current study, the inhibitory effect of BmKCT on glioma growth was observed in vivo using the glioma/SD rat model. Furthermore, BmKCT prevented the metastasis of glioma cells in vivo. Moreover, biodistribution experiments with (l3l)I-labeled or Cy5.5-conjugated BmKCT revealed that BmKCT selectively targeted the glioma in situ. Our data suggest that BmKCT could be exploited as a potential therapeutic for glioma diagnosis and therapy.
[Show abstract][Hide abstract] ABSTRACT: C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.
Full-text · Article · Oct 2008 · Biochemistry (Moscow)
[Show abstract][Hide abstract] ABSTRACT: Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, NIH3T3 pcDNA3-DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP were protected from C-mediated damage and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack, which was compared with either DAF or MCP alone. These results suggest that the dicistronic vector could improve the efficiency of multi-gene delivery and benefit the synergic effect of human membrane complement regulatory proteins DAF and MCP.
No preview · Article · Mar 2008 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology
[Show abstract][Hide abstract] ABSTRACT: Since its discovery, green fluorescence protein (GFP) has been used as a reporter in a broad range of applications, including the determination of gene expresion in diverse organisms, and subcellular protein localization. pEGFP-N1 is a eukayotic expression vector encoding EGFP, the MCS of which locates at the N terminus of EGFP. In this study, the cDNA sequence of scorpion toxin BmKK2 was inserted into the XhoI-HindIII cut of pEGFP-N1 to construct a toxin-EGFP fusion gene (named pEGFP-BmKK2). Fluorescence imaging revealed that HEK 293T cells that were transfected by pEGFP-BmKK2 emitted green fluorescence. Transcription of pEGFP-BmKK2 was confirmed by RT-PCR. However, western blotting analysis showed that the transfected HEK 293T cells expressed mostly EGFP, but little toxin-EGFP fusion protein, implying that pEGFP-N1 cannot be used as a fusion expression vector for subcellular protein localization for the BmKK2 gene. Consequently, two modified recombinant vectors (pEGFP-BmKK2-M1 and pEGFP-BmKK2-M2) were constructed based on pEGFP-BmKK2. This greatly improved the expression of toxin-EGFP fusion protein from pEGFP-BmKK2-M2.
[Show abstract][Hide abstract] ABSTRACT: SARS-CoV spike (S) protein-mediated cell fusion is important for the viral entry mechanism and identification of SARS-CoV entry inhibitors. In order to avoid the high risks involved in handling SARS-CoV and to facilitate the study of viral fusion mechanism, we established the cell lines: SR-COS7 cells that stably express both SARS-CoV S protein and red fluorescence protein, R-COS7 cells that stably express red fluorescence protein, and AG-COS7 cells that stably express both ACE2 and green fluorescence protein, respectively. When SR-COS7 cells or R-COS7 cells were cocultured with AG-COS7 cells, syncytia with yellow fluorescence were conveniently observed after 12 h in SR-COS7 cells plus AG-COS7 cells, but not in R-COS7 cells plus AG-COS7 cells. The cell-to-cell fusion efficiency was simply determined for quantitative analysis based on the number of syncytium detected by flow cytometry. Such new cell-to-cell fusion model was further assessed by the potent HR2 peptide inhibitor, which led to the obvious decrease of the cell-to-cell fusion efficiency. The successful fusion and inhibition of cell-based binding assay shows that it can be well used for the study of SARS-CoV entry and inhibition.
Full-text · Article · Sep 2006 · International Union of Biochemistry and Molecular Biology Life
[Show abstract][Hide abstract] ABSTRACT: The human complement regulatory proteins (hCRPs) decay accelerating factor (DAF/CD55) and protectin CD59 transfected into non-human cells could confer protection against human complement. The combination of DAF and CD59 would be an effective strategy to help overcome host complement-induced hyperacute rejection in xenotransplantation. We constructed a dicistronic mammalian expression vector pcDNA3-CD59IRESDAF by using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). RT-PCR, Western blotting and immunofluorescence microscopic analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hCD59 and hDAF on the surface of NIH/3T3 cells transfected stably with pcDNA3-CD59IRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and CD59 proteins could provide more significant protection against complement-mediated cytolysis than either hCD59 or hDAF alone. These results suggest that IRES containing polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery and that the construct pcDNA3-CD59IRESDAF vector has potential therapeutic value for effectively controlling complement activation and for preventing hyperacute rejection in clinical gene therapy.
No preview · Article · Oct 2005 · International Journal of Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Aug 2005 · International Union of Biochemistry and Molecular Biology Life
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Aug 2005 · Journal of biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: Based on the reported cDNA sequences of , the genes encoding toxin and 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 and . Using cDNA sequence of as probe, southern hybridization of BmK genome total DNA was performed. The result indicates that is multicopy genes or belongs to multiple gene family with high homology genes. The similarity of -toxin gene sequences and southern hybridization revealed the evolution trace of -toxins: -toxin genes evolve from a common progenitor, and the genes diversity is associated with a process of locus duplication and gene divergence.
[Show abstract][Hide abstract] ABSTRACT: Computational methods are employed to simulate interaction of scorpion toxin ScyTx in complex with the small conductance calcium-activated potassium channel rsk2. All of available 25 structures of ScyTx in the Protein Data Bank determined by NMR were considered for improving performance of rigid protein docking of ZDOCK. Four main binding modes were found among a large number of predicted complexes by using clustering analysis, screening with expert knowledge, energy minimization, and molecular dynamics simulations. The quality and validity of the resulting complexes were further evaluated by molecular dynamics simulations with the generalized Born solvation model and by calculation of relative binding free energies with the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) in the AMBER 7 suit of programs. The complex formed by the 22nd structure of the ScyTx and rsk2 channel was identified as the most favorable complex by using a combination of computational methods, which contain further introduction of flexibility without restraining residue side chain. From the resulted spatial structure of the ScyTx and rsk2 channel, ScyTx associates the mouth of the rsk2 channel with alpha-helix rather than beta-sheet. Structural analysis first revealed that Arg(13) played a novel and vital role of blocking the pore of the rsk2 channel, whose role is remarkably different from that of highly homologous scorpion toxin P05. Between the interfaces in the ScyTx-rsk2 complex, strong electrostatic interaction and hydrogen bonds exist between Arg(13) of ScyTx and Gly-Tyr-Gly-Asp sequential residues located in the four symmetrical chains of the pore region. Simultaneously, five hydrogen bonds between Arg(6) of ScyTx and Asp(341)(C), Val(366)(C), and Pro(367)(C), and electrostatic interaction between Arg(6) of ScyTx and Asp(364)(B) and Asp(341)(C) are also found by structural analysis. In addition, His(31) located at the C-terminal of ScyTx is surrounded by Val(342)(A), Asp(364)(A), Met(365)(A), Pro(367)(B), and Asn(366)(B) within a contact distance of 4.0 A. These simulation results are in good agreement with experimental data and can effectively explain the binding phenomena between ScyTx and the potassium channel at the level of molecular spatial structure. The consistency between results of molecular modeling and experimental data strongly suggests that our spatial structure model of the ScyTx-rsk2 complex is reasonable. Therefore, molecular docking combined with molecular dynamics simulations followed by molecular mechanics Poisson-Boltzmann surface area analysis is an attractive approach for modeling scorpion toxin-potassium channel complexes a priori for further biological studies.
Preview · Article · Aug 2004 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: BmTXKbeta, 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-BmTXKbeta protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKbeta fusion protein released an approximate 6.5kDa protein which was the expected size for correctly processed. About 2mg purified recombinant BmTXKbeta protein (rBmTXKbeta) was produced from 1l bacterial culture, using this expression and purification system. The function of rBmTXKbeta was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKbeta inhibited the transient outward current (I(to)) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKbeta prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.
[Show abstract][Hide abstract] ABSTRACT: CD59 and membrane cofactor protein (MCP, CD46) are widely expressed cell surface glycoproteins that protect host cells from the effect of homologous complement attack. cDNAs encoding human CD59 and MCP cloned from Chinese human embryo were separately transfected into NIH/3T3 cells resulting in the expression of human CD59 and MCP protein on the cell surface. The functional properties of expressed proteins were studied. When the transfected cells were exposed to human serum as a source of complement and naturally occurring anti-mouse antibody, they were resistant to human complement-mediated cell killing. However, the cells remained sensitive to rabbit and guinea pig complement. Human CD59 and MCP can only protect NIH/3T3 cells from human complement-mediated lysis. These results demonstrated that complement inhibitory activity of these proteins is species-selective. The cDNAs of CD59 and MCP were also separately transfected into the endothelial cells (ECs) of the pigs transgenic for the human DAF gene to investigate a putative synergistic action. The ECs expressing both DAF and MCP proteins or both DAF and CD59 proteins exhibited more protection against cytolysis by human serum compared to the cells with only DAF expressed alone.
[Show abstract][Hide abstract] ABSTRACT: K+ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K+ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca2+-activated K+ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length
cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA,
which is highly conserved in scorpion Na+ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation.
The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide
of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its
length is significantly different from that of the determined scorpion Na+ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined
by primary structure analysis.
No preview · Article · Jan 2000 · Chinese Science Bulletin
[Show abstract][Hide abstract] ABSTRACT: Five full-length cDNAs encoding the precursors of two 'short chain' scorpion non-toxic peptides active on Ca(2+)-activated K(+) channels (BmP02 and BmP03) and two novel putative long chain K(+) channel-blocking peptides (named BmTXKbeta and BmTXKbeta2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKbeta2 showed a high similarity with AaTXKbeta, while BmTXKbeta was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKbeta expands the scorpion long chain K(+) channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an alpha-helix and a three-stranded beta-sheet.