间斑寇蛛毒素的蛋白质化学与蛋白质组学研究新进展

间斑寇蛛Latrodectus tredecimguttatus俗称"黑寡妇"蜘蛛。其毒素不仅存在于毒腺中,而且存在于其身体的其他部分、卵粒甚至新生幼蛛体内。研究间斑寇蛛毒腺和毒腺外材料中的毒素成分,探明它们之间的异同、进化关系和生物学作用,具有重要的理论和实际意义。现代蛋白质化学和蛋白质组学技术的发展为间斑寇蛛蛋白质和多肽毒素的研究提供了有效手段,从而可以同时从单一纯化蛋白质和组学的层面探究毒素作用的分子基础和作用机制。到目前为止,间斑寇蛛毒素的蛋白质化学与蛋白质组学研究已取得一定的进展,但相关研究尤其是毒腺外材料来源的毒素研究还有待进一步深入。     

Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus]

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.     

Physiological and biochemical analysis of L. tredecimguttatus venom collected by electrical stimulation

The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 microg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.     

间斑寇蛛 L.tredecimguttatus粗毒抗血清制备及其活性分析

间斑寇蛛Latrodectus tredecimguttatus是世界上毒性最强的蜘蛛之一,其毒液中含有许多大分子物质,可用于制备抗血清。将粗毒过滤除菌后作为抗原,加弗氏佐剂背部皮下免疫豚鼠。加强免疫3次后经检测抗血清效价达到1：8,再次加强免疫后断颈处死豚鼠放血,分离血清,经饱和硫酸铵分级沉淀及阴离子交换柱层析纯化后,采用westernblot法检测抗体所在峰。离体大鼠输精管电刺激收缩反应实验及活体动物给药实验均表明抗体对毒素具有明显的中和作用。特异性间斑寇蛛毒素抗血清为治疗间斑寇蛛咬伤奠定了基础。     

间斑寇蛛粗毒液的生理生化分析及两种采毒方法的比较

利用两种不同方法采集间斑寇蛛（Latrodectus tredecimguttatus）毒液并对其进行理化和生物学性质的比较分析。结果显示,粗毒液中的蛋白质成分主要是相对分子质量较大（>104）的酸性蛋白质。与毒囊粗毒液相比,电刺激粗毒液中相对分子质量在105左右的蛋白质含量明显高于毒囊粗毒液中的含量,而两者中相对分子质量较小（<104）的蛋白质与多肽的组成非常相似。电刺激粗毒冻干粉和毒囊粗毒冻干粉对小白鼠的LD50值分别为(0.16±0.03) mg/kg和(0.39±0.05)mg/kg体重;对美洲蜚蠊（Periplaneta Americana）的LD50值分别为1.87 μg/g和2.32 μg/g。在浓度为3.2×10-6g/mL时,电刺激粗毒冻干粉能在（25.0±2.2） min内完全阻断小鼠膈神经-膈肌标本的神经肌肉接头传递;毒囊粗毒冻干粉在浓度为6×10-6g/mL时的完全阻断时间为（23.3±2.2） min;粗毒液中的低相对分子质量（<104）部分在10-4g/mL浓度下对标本的神经肌肉接头传递无明显影响。上述结果表明,间斑寇蛛毒液是一种富含大分子量蛋白质的混合物;哺乳动物神经毒性主要基于其中的大的相对分子质量酸性蛋白质成分,而不是低的相对分子质量的多肽;电刺激粗毒液中的活性成分与毒囊粗毒液中的相似,但含量高于毒囊粗毒液。     

Interaction of alpha-(125)latrocrustotoxin with nerve cell membranes from the river crab Astacus astacus]

alpha-Latrocrustatoxin, the crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus was radioactively labelled with Bolton-Hunter reagent to the specific activity of 160 Ci/mmol with retention of the biological activity. A highly specific binding of radioactive toxin on plasmatic membranes from the crayfish Astacus astacus nerve cells with Bmax = 0.04 pmol binding toxin/mg membrane protein and Kd = 0.7 x 10(-10) M was demonstrated.     

Extraction and protein component analysis of venom from the dissected venom glands of Latrodectus tredecimguttatus

Black widow spiders (genus Latrodectus) have attracted increasing attention due to frequently reported human injuries caused by them and the potential applications of biologically active components in their venoms. Although a number of studies have described the biological properties and structures of several venomous proteins such as latrotoxins, a comprehensive analysis of protein component of the venom from the spider is not available. We used combinative proteomic strategies to assess the protein components of the crude venom collected from Latrodectus tredecimguttatus by extracting the dissected venom glands. The experiments demonstrated that the crude venom of L. tredecimguttatus has a high abundance of acidic proteins with molecular masses greater than 15 kDa, and the content of proteins and peptides of below 15 kDa is low. 86 unique proteins were identified, part of which were contaminations of cellular components during the extraction, determined in comparison with venom obtained by electrostimulation. Except for members of latrotoxin family that were commonly considered as the primary toxic components of the venom, several other special enzymes and proteins were detected such as protease, phosphatase, lysozyme, inhibitory protein, and so on. These protein components, particularly the proteases, were speculated to play important roles in the action of L. tredecimguttatus venom.     

alpha-Latrotoxin as an instrument for studying neurosecretions

alpha-Latrotoxin, a presynaptic toxin from black widow spider venom Latrodectus mactans tredecimguttatus, triggers exocytosis in a variety of neurosecretory cells both in the presence and absence of calcium in the medium. The toxin interacts with two types of membrane the receptors which belong to different families of neuronal proteins and have different structures. Calcium-dependent receptor of alpha-latrotoxin is identified as neurexin I alpha and belongs to the family of neurexins. This family is selectively expressed in nerve tissue. The calcium-independent receptor of alpha-latrotoxin belongs to the family of G-protein-coupled receptors and proteins which homologous to it are found in heart, lung, kidney and spleen tissues. As a result of alpha-latrotoxin interaction with membrane receptor in the calcium medium the toxin forms the ionic channels in plasmalemma and enhances its calcium permeability. The effects of alpha-latrotoxin on exocytosis in the calcium and calcium-free media and question concerning coupling of channel-forming and secretogenic properties of alpha-latrotoxin are discussed.     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

Study of the receptor for black widow spider neurotoxin. I. Characteristics of membrane-bound and solubilized receptors from the bovine brain

Iodine-125 labelled alpha-latrotoxin from the venom of Central Asia black widow spider Latrodectus mactans tredecimguttatus binds specifically to the bovine brain membrane receptor producing a stable slowly dissociating complex with Kd = 1.6 x 10(-10) M and Bmax = 0.5 pmol/mg protein. Treatment of the complex with alkaline high-salt buffer induces reversible dissociation of the bound toxin. The antitoxin polyclonal antibody does not increase the dissociation rate of the bound toxin. Wheat germ lectin as well as concanavalin A inhibit the toxin binding to the membrane receptor. The receptor is solubilized with ionic and non-ionic detergents, and methods of latrotoxin binding assay are developed. The solubilized receptor is shown to retain high affinity to toxin, its binding activity being stable but critically dependent on the presence of calcium ions. Chromatographic properties of the receptor suggest its glycoprotein nature.     

Activities against hemostatic proteins and adrenal gland ultrastructural changes caused by the brown widow spider Latrodectus geometricus (Araneae: Theridiidae) venom

Brown widow spider (BrWS) (Latrodectus geometricus) venom produces intense systemic reactions such as cramps, harsh muscle nociceptive, nauseas, vomiting and hypertension. The proposed pathogenic mechanisms resulting in these accidents have principally been damages occurring at the nervous system. However, it is suspected that there is also damage of the adrenal glands, as a result of the experimental animal's clinical manifestations, which developed symptoms compatible with acute adrenal insufficiency. We have currently found that the adrenal gland is damaged by this venom gland homogenates (VGH) producing severe alterations on cortex cells resulting in death by acute adrenal insufficiency. In general, the ultrastructural study on the glands of mice under transmission electronic microscopy observations showed alterations in the majority of the intracellular membranes within 3 to 24 h. BrWSVGH also showed specific actions on extracellular matrix proteins such as fibronectin, laminin and fibrinogen. In addition, zymogram experiments using gelatin as substrates detected gelatinolytic activity. The molecular exclusion fractionation of crude BrWSVGH resulted in 15 fractions, of which F1 and F2 presented α/β-fibrinogenase and fibronectinolytic activities. Fractions F6, F14 and F15 showed only α-fibrinogenase activity; in contrast, the gelatinolytic action was only observed in fraction F11. Only metalloproteinase inhibitors abolished all these proteolytic activities. Our results suggest that adrenal cortex lesions may be relevant in the etiopathogenesis of severe brown widow spider envenoming. To our knowledge, this is the first report on adrenal gland damages, fibrinogenolytic activity and interrelations with cell-matrix adhesion proteins caused by L.geometricus VGH. The venom of this spider could be inducing hemostatic system damages on envenomed patients.     

间斑寇蛛(L.tredecimguttatus)室内饲养及活体采毒方法的初步探讨

间斑寇蛛Latrotectus tredecimguttatus是目前已知毒性最强的蜘蛛之一,为了解决间斑寇蛛毒素研究中毒素来源有限的问题,本实验室开展了间斑寇蛛的室内人工饲养与采毒方法的研究,着重探索影响室内饲养间斑寇蛛生长发育、成活率及繁殖力的主要因素及活体采毒方法.结果表明,温度、湿度和食物种类等多种因素都影响湖南一带室内饲养间斑寇蛛的效果,但相对而言,空气的相对湿度是最重要的因素.与同批次的雌、雄蛛交配比较,利用前批次雌蛛与相隔30天左右的后批次雄蛛交配更有利于产卵率的提高.间斑寇蛛幼蛛度过适当长度的"卵袋期"对于以后的成活与生长发育,尤其是早期的成活与生长发育来说是必要的.尽管有多种方法可用来进行间斑寇蛛的活体采毒,但电刺激采毒法是最佳的活体采毒方法.     

The channel-forming component of the Theridiidae spider venom neurotoxins

It is known that Steatoda (Lityphantes) paykulliana and Latrodectus mactans tredecimguttatus spider venoms are toxic to mammals and insects. These venoms act presynaptically eliciting massive release of transmitters. They also form channels in bilayer lipid membranes (BLM) that are selective for cations. Venoms of both spider species were fractionated by gel filtration on a Sephadex G-100 column. The fraction obtained were tested on neuromuscular preparations of frog and locust and on BLM. A fraction of low molecular weight components (about 5000 daltons and less) was disclosed. This fraction showed presynaptic and channel-forming effects similar to those of crude venoms and of high molecular weight toxin fractions, obtained simultaneously from these venoms. It was shown that channels formed in BLM by crude venoms and its different fractions are identical. Also, it was found that the low molecular weight channel-forming component is a construction element of high molecular weight toxins. On the basis of data obtained a toxin structure model of the Theridiidae family spider venoms was proposed.     

A crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus

A method of the isolation of a crustacea-specific neurotoxin from the venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and hydrophobic chromatography on Phenyl-Superose column has been developed. LD50 of the toxin has been elucidated.     

Isolation and properties of insect-specific neurotoxins from venoms of the spider Lactodectus mactans tredecimguttatus

A method has been developed for isolating five insect-specific neurotoxins and alpha-latrotoxin from venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and chromatography on hydroxylapatite column. LD 50 of all the toxins are determined.     

Identification of the neuronal protein with antigenic properties of latrotoxin

Monospecific antibodies against neurotoxin from the black widow spider venom (alpha-latrotoxin) have been used to identify a neuronal protein (s) with latrotoxin-like epitopes. As determined by ELISA, the protein is localized in cytoplasmic fraction of bovine brain. It was partially purified by anion-exchange chromatography and preparative SDS-electrophoresis. Immunoblotting data indicate that this neuronal acidic protein has molecular mass of ca. 100 kDa.     

Proteomic and peptidomic characterization of the venom from the Chinese bird spider, Ornithoctonus huwena Wang

The bird spider Ornithoctonus huwena Wang is a very venomous spider in China. Several compounds with different types of biological activities have been identified previously from the venom of this spider. In this study, we have performed a proteomic and peptidomic analysis of the venom. The venom was preseparated into two parts: the venom proteins with molecular weight (MW) higher than 10,000 and the venom peptides with MW lower than 10 000. Using one-dimensional gel electrophoresis (1-DE), two-dimensional gel electrophoresis (2-DE), and mass spectrometry, 90 proteins were identified, including some important enzymes, binding proteins, and some proteins with significant biological functions. For venom peptides, a combination of cation-exchange and reversed-phase chromatography was employed. More than 100 components were detected by mass spectrometry, and 47 peptides were sequenced by Edman degradation. The peptides display structural and pharmacological diversity and share little sequence similarity with peptides from other animal venoms, which indicates the venom of O. huwena Wang is unique. The venom peptides can be classified into several superfamilies. Also it is revealed that gene duplication and focal hypermutation have taken place during the evolution of the spider toxins.     

Human female bladder and its noncholinergic contractile function

The response of human female detrusor muscle to field stimulation at varying voltages, durations, and frequencies was studied in vitro. In addition, the effects of adrenergic and cholinergic agonists and antagonists, and various nerve toxins were studied. Beta-adrenergic receptors were found in detrusor muscle but no significant adrenergic innervation was seen; no alpha-adrenergic receptors were seen. Atropine, scorpion venom, tetrodotoxin, beta bungarotoxin and hemicholinium were found to inhibit bladder contraction at short-pulse durations and low frequencies by approximately 50%. Black widow spider venom was seen to abolish bladder contractions entirely. It is concluded that acetylcholine is the neurotransmitter responsible for approximately 50% of bladder contraction. The remaining 50% would seem to be noncholinergic and not dependent on fast sodium channels for transmission of excitation, but would seem to be due to a structure with a short-membrane time constant, such as nerve, and is sensitive to black widow spider venom.     

Recruitment and diversification of an ecdysozoan family of neuropeptide hormones for black widow spider venom expression

Venoms have attracted enormous attention because of their potent physiological effects and dynamic evolution, including the convergent recruitment of homologous genes for venom expression. Here we provide novel evidence for the recruitment of genes from the Crustacean Hyperglycemic Hormone (CHH) and arthropod Ion Transport Peptide (ITP) superfamily for venom expression in black widow spiders. We characterized latrodectin peptides from venom gland cDNAs from the Western black widow spider (Latrodectus hesperus), the brown widow (Latrodectus geometricus) and cupboard spider (Steatoda grossa). Phylogenetic analyses of these sequences with homologs from other spider, scorpion and wasp venom cDNAs, as well as CHH/ITP neuropeptides, show latrodectins as derived members of the CHH/ITP superfamily. These analyses suggest that CHH/ITP homologs are more widespread in spider venoms, and were recruited for venom expression in two additional arthropod lineages. We also found that the latrodectin 2 gene and nearly all CHH/ITP genes include a phase 2 intron in the same position, supporting latrodectin's placement within the CHH/ITP superfamily. Evolutionary analyses of latrodectins suggest episodes of positive selection along some sequence lineages, and positive and purifying selection on specific codons, supporting its functional importance in widow venom. We consider how this improved understanding of latrodectin evolution informs functional hypotheses regarding its role in black widow venom as well as its potential convergent recruitment for venom expression across arthropods.