蝎氯毒素结构、功能与应用研究

蝎氯毒素是一类能特异阻断神经胶质瘤氯电流的短链蝎毒素。它们具有高度的同源性、保守的基因序列与相似的3-D结构。根据其结构与功能的关系,推测它们可能有相似的药理功能。其中,Chlorotoxin(Cltx)能与神经胶质瘤细胞特异相互作用,并抑制其侵袭与转移。     

蝎毒素多肽致/抗痛的靶离子通道机制

疼痛调控是多通道、多受体和多系统介导的复杂生命医学热点问题。诸多靶通道特异性工具药的普遍缺乏,致使其机制解析和临床诊疗转化任重道远。蝎毒是多种毒素多肽的集合体,用于捕食和防御天敌,蛰伤诱致长时程炎症和疼痛。通常认为,类α长链肽类蝎毒素作用于钠通道,延缓钠通道失活,产生致痛效应,而类β长链多肽蝎毒素则相反。短链肽类蝎毒素作用于钾通道、氯通道和钙通道,呈现疼痛相关的功能多样化。因此,开发应用极具潜力的靶向蝎毒素,是解析疼痛调制新机制的重要工具分子。现对蝎毒素与疼痛相关离子通道互作的工作做一综述,并探讨蝎毒素多肽致/抗痛的分子功能多样性。     

蝎β型昆虫毒素的结构及其与钠通道作用机制

蝎毒素是蝎为防卫的需要而产生的一系列活性短肽.其中蝎昆虫特异性毒素可特异性结合并调控昆虫可兴奋细胞膜上的钠离子通道,是研究离子通道结构与功能的首选探针,并在转基因抗虫植物及生物杀虫剂研究方面具有潜在的应用价值.本文对蝎β型昆虫毒素的结构与功能及其对钠离子通道的作用方式和β毒素的电压传感器捕获(voltage sensor-trapping)模型做一综述,为进一步揭示蝎β毒素的结构与功能的关系和在农作物抗虫领域的应用提供依据.     

肽类毒素(蜘蛛、蝎、芋螺)的三维结构

多肽类毒素研究是目前毒素研究的一个重点,对多肽类毒素的三维结构的研究是了解其结构与功能关系的重要基础.对蜘蛛、蝎以及芋螺这3类代表性的有毒动物的多肽类毒素在结构研究方面的进展及其三维结构的特点进行了介绍.其中,蜘蛛毒素多肽分子的结构主要发现有ICK模体(Inhibitor Cystine Knot motif)和D DH模体(disulfided-irectedh-airpin)两类,蝎毒素中长链肽类毒素分子和短链肽类毒素分子的结构明显不同,前者以CSα/β结构模体(Cyss-tabilizedα/βfold m otif)为主,后者则以α/β脚手架结构模体(α/βscaffoldm otif)为主.相对于蜘蛛和蝎而言,芋螺肽类毒素分子的三维结构则表现得更为复杂多样.     

一个东亚钳蝎蝎毒素BmTXKβ基因的克隆及其基因表达调控

从东亚钳蝎 (ButhusmartensiiKarsch ,BmK)毒腺组织cDNA文库中分离的长链钾通道毒素BmTXKβcDNA序列 ,克隆了BmTXKβ基因组序列 .BmTXKβ基因含有一个长度为 886bp的内含子 ,定位于BmTXKβ成熟肽中 ,与其它蝎毒素基因内含子定位于信号肽的基因结构不同 .并且 ,BmTXKβ基因的内含子特征也与其它蝎毒素基因不同 .研究结果从基因水平上证实了BmTXKβ是一个新的蝎毒素样肽 .以BmTXKβcDNA序列为探针与蝎基因组DNASouthern杂交出现 2条特异性杂交带 .杂交结果为蝎毒素基因可能通过DNA重排、多拷贝或多基因家族来调控基因表达提供了证据 .     

一种新的蝎毒素BmTXKβ的基因组结构及分析

根据从东亚钳蝎(Buthus Martensii Karsch,BmK)毒腺cDNA文库中筛选得到的长链钾离子通道毒素BmTXKβ基因序列设计引物,采用Vectorette方法克隆其5′及3′侧翼区。结果表明：BmTXKβ在编码信号肽的基因序列中有—大于997bp的内含子,并且其成熟肽中也有一个886bp内含子,而目前已知的其他蝎毒素只在信号肽中有一个内含子或没有内含子。BmTXKβ基因组结构的独特性更进一步证实BmTXKβ是一个新的长链钾离子通道毒素成员。     

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     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

蝎毒素基因分子生物学研究进展

介绍了克隆蝎毒素cDNA和基因组基因的策略以及蝎毒素cDNA的结构和毒素蛋白前体的翻译后加工过程,同时综述了蝎毒素基因组基因的组织结构及其mRNA前体的加工以及重组蝎毒素基因表达的研究进展.     

作用于钾离子通道蝎毒素的结构特征及活性表面研究进展

对作用于钾离子通道的蝎毒素的空间结构特点进行了简要归纳,发现高含量碱性残基在不同结构单元广泛分布而少量酸性残基特征性分布等新特点。蝎毒素活性表面研究进展表明,利用空间结构的分子模建结合残基突变是确定活性表面的有效方法。基于少量酸性残基特征分布与活性表面取向的相关性,提出酸性残基为活性调节残基的新观点和简单的“拇指”规则预测钾毒素活性表面的方法,从而可望加速蝎毒素的结构与功能关系研究。     

Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.     

Effects of Mitochondrial Toxins on the Brain Amino Acid Concentrations

In the pathogenesis of Parkinson’s disease and Huntington’s disease excitotoxicity may play an important role. The common toxin model for Parkinson’s disease is MPTP, while for Huntington’s disease it is 3-NP. These toxins inhibit the mitochondrial respiratory chain, resulting in an energy deficit. In the central nervous system, the amino acids act as neurotransmitters and neuromodulators. The energy deficit caused by these neurotoxins may alter the concentrations of amino acids. Thus, it can be claimed that the aminoacidergic neurotransmission can be changed by neurotoxins. To test this hypothesis we studied the amino acid concentrations in different brain regions following MPTP or 3-NP administration. The two toxins were found to produce similar changes. We detected marked decreases in most of the amino acid concentrations in the striatum and in the cortex, while the levels in the cerebellum increased significantly. The decreased amino acid levels can be explained by the reduced levels of ATP produced by these neurotoxins. In the cerebellum, where there is no detectable ATP loss, the elevated amino acid levels may reflect a compensation of the altered neurotransmission.     

Functional discrimination of sea anemone neurotoxins using 3D-plotting

One of the most important goals in structural biology is the identification of functional relationships among the structure of proteins and peptides. The purpose of this study was to (1) generate a model based on theoretical and computational considerations among amino acid sequences within select neurotoxin peptides, and (2) compare the relationship these values have to the various toxins tested. We employed isolated neurotoxins from sea anemones with established specific potential to act on voltage-dependent sodium and potassium channel activity as our model. Values were assigned to each amino acid in the peptide sequence of the neurotoxins tested using the Number of Lareo and Acevedo algorithm (NULA). Once the NULA number was obtained, it was then plotted using three dimensional space coordinates. The results of this study allow us to report, for the first time, that there is a different numerical and functional relationship between the sequences of amino acids from sea anemone neurotoxins, and the resulting numerical relationship for each peptide, or NULA number, has a unique location in three-dimensional space.     

蝎短肽链神经毒素研究进展

对蝎短肽链神经毒素结构与功能研究进展作了简要的论述,蝎毒中富含短肽链神经毒素,至今已经分离纯化到60多种,它们的大小介于28－41个氨基酸残基之间,分子中含有3－4对二硫键,空间结构紧密,这些毒素可以特异性地与K＋,Cl-和Ca2 等离子通道相结合,由于它们对离子通道的选择性,这些毒素在药理学和神经生物学中已经得到了广泛的应用。     

Two Novel α-Neurotoxins Isolated from Taiwan Cobra: Sequence Characterization and Phylogenetic Comparison of Homologous Neurotoxins

Two novel postsynaptic neurotoxins (-neurotoxins) isolated and purified from the Taiwan cobra venom (Naja naja atra) possess distinct primary sequences and different neurotoxicities as compared with the most abundant and lethal component in the venom, i.e., cobrotoxin characterized before from the same venom. The complete sequences of two neurotoxin analogues were determined by N-terminal Edman degradation and comparison of amino acid compositions of proteolytic toxin fragments with other homologous toxins of known sequences. The short-chain neurotoxin consists of 61 amino acid residues with eight conserved cysteine residues and is found to show 78% sequence identity with cobrotoxin. The other toxin, consisting of 65 residues with ten cysteines, belongs to the family of long-chain neurotoxins. It is the first long-chain -neurotoxin reported from the Taiwan cobra. The lethal toxicities of these two novel neurotoxins were much lower than cobrotoxin, albeit with close structural homology among the three toxins in terms of their primary sequences and tertiary structure predicted by homology modeling. Multiple sequence alignment and comparison coupled with construction of a phylogenetic tree for various -neurotoxins of Naja and closely related genuses have established that all nicotinic -neurotoxins present in the snake family of Elapidae are closely related to each other, presumably derived from an ancestral polypeptide by gene duplication and subsequent multiple mutational substitutions.     

Amino acid sequences of two novel long-chain neurotoxins from the venom of the sea snake Laticauda colubrina.

From the venom of a population of the sea snake Laticauda colubrina from the Solomon Islands, a neurotoxic component, Laticauda colubrina a (toxin Lc a), was isolated in 16.6% (A280) yield. Similarly, from the venom of a population of L. colubrina from the Philippines, a neurotoxic component, Laticauda colubrina b (toxin Lc b), was obtained in 10.0% (A280) yield. The LD50 values of these toxins were 0.12 microgram/g body wt. on intramuscular injection in mice. Toxins Lc a and Lc b were each composed of molecules containing 69 amino acid residues with eight half-cystine residues. The complete amino acid sequences of these two toxins were elucidated. Toxins Lc a and Lc b are different from each other at five positions of their sequences, namely at positions 31 (Phe/Ser), 32 (Leu/Ile), 33 (Lys/Arg), 50 (Pro/Arg) and 53 (Asp/His) (residues in parentheses give the residues in toxins Lc a and Lc b respectively). Toxins Lc a and Lc b have a novel structure in that they have only four disulphide bridges, although the whole amino acid sequences are homologous to those of other known long-chain neurotoxins. It is remarkable that toxins Lc a and Lc b are not coexistent at the detection error of 6% of the other toxin. Populations of Laticauda colubrina from the Solomon Islands and from the Philippines have either toxin Lc a or toxin Lc b and not both of them.     

Peptides of arachnid venoms with insecticidal activity targeting sodium channels

Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.     

Molecular mechanisms of neurotoxin action on voltage-gated sodium channels

Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.     

Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini.

From the venom of a sea snake Astrotia stokesii three neurotoxic components, toxins Astrotia stokesii a, b and c were isolated in 40, 15 and 5% yield by weight respectively of the whole venom. Their LD50 values for 20g mice were 0.13, 0.096 and 0.098 microgram/g body wt. respectively and accounted for almost all the lethal activity of the venom. Their amino acid sequences were determined. Astrotia stokesii a was composed of 60 amino acid residues with nine half-cystine residues and was quite homologous to other sea-snake short-chain neurotoxins in its amino acid sequence. Toxins Astrotia stokesii b and c were composed of 70 and 72 amino acid residues respectively with 10 half-cystine residues. They are the first long-chain neurotoxins with high activity isolated from sea-snake venoms. The C-terminal carboxy groups of toxins b and c were found to be amidated; the amidation is known for some polypeptides, but is novel for a protein. The amide group may make a hydrogen-bond with glutamic acid-39, which replaces a lysine that has so far been found invariably in long-chain neutrotoxins. Astrotia stokesii b and c are also novel in having phenylalanine-25 and isoleucine- or valine-42. The ordinary Tyr-Glu pair, which is observed in X-ray structure [Low, Preston, Sato, Rosen, Searl, Rudko & Richardson (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2991-2994] and n.m.r.study [Inagaki, Tatsumi, Miyazawa, Hori & Tamiya (1977) Abstr. Int. Congr. Pure Appl. Chem. 26th, p. 336] on erabutoxins may be replaced by a hydrophobic pair. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 5009o (30 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B1, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Functional duality and structural uniqueness of depressant insect-selective neurotoxins

Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.