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一种源自北非蝎的抗昆虫毒素Am IT的结构与功能鉴定

Naoual Oukkache, Rachid ElJaoudi, Fatima Chgoury, Marisa Teixeira Rocha, Jean-Marc Sabatier

摩洛哥巴斯德研究所毒液和毒素实验室，卡萨布兰卡 20360，摩洛哥；布坦坦研究所毒液学实验室爬虫学部，圣保罗 05503-900，巴西；拉巴特研究所医学和药学系药理毒理实验室，拉巴特，摩洛哥；艾克斯-马赛大学全国保健与医学研究所(INSERM)，马赛 13288，法国

摘要

本研究主要分离和鉴定了一种源自北非蝎被称为Am IT的新毒素。详细分析了Am IT 对昆虫神经索钠电流的调节效应。Am IT经凝胶过滤纯化后通过C18反相高效液相色谱法纯化。采用雄性德国蟑螂(德国小蠊)的幼虫和C57/BL6小鼠进行Am IT体内毒性分析。Am IT与两种β毒素的竞争实验则采用125I修饰的毒素通过与大鼠脑突触体膜的放射免疫法测定。Am IT的氨基酸序列通过Edman降解法测定。在结合实验中，Am IT被观察到与另一种源自北非蝎的毒素AaH IT4存在竞争效应。而且它可以被β型毒素的抗体识别，由此说明它与β型毒素(或相关毒素家族)的结构相似。新毒素显示了双重的活性，它既能与抗哺乳动物毒素在结合实验中竞争，又能促使昆虫躯体产生收缩反应。该毒素能够竞争携带放射标记的β毒素Css IV与大鼠突触体膜钠通道的结合。通过氨基酸序列分析，Am IT显示出与AaH IT4有92%的同源性。以上结果说明，Am IT不但显示了隶属于旧大陆的抗昆虫毒素的性质，表现为结合于昆虫的钠通道；而且它与隶属于新大陆的抗哺乳动物毒素类似，表现为对小鼠的毒性。因此，Am IT与AaH IT4在结构与功能上是极为类似的。

关键词： 北非蝎毒; 蝎毒素; Am IT; β毒素; 抗哺乳动物毒素; 抗昆虫毒素; 钠通道; 毒性; 从头测序; Edman降解法

分类号：Q965.9;Q71

Characterization of Am IT, an anti-insect β-toxin isolated from the venom of scorpion Androctonus mauretanicus

Naoual Oukkache, Rachid ElJaoudi, Fatima Chgoury, Marisa Teixeira Rocha, Jean-Marc Sabatier

Laboratory of Venoms and Toxins, Pasteur Institute of Morocco, Casablanca 20360, Morocco； Laboratory of Venoms-Herpetology, Institute Butantan, Sao Paulo 05503-900, Brazil； Laboratory of Pharmacology and Toxicology, Faculty of Medicine and Pharmacy, Rabat-Institus, Rabat, Morocco； Laboratory INSERM UMRs 1097, Aix-Marseille University, Science and Technology Park of Luminy, Marseille 13288, France

Abstract

In the present study, a ‘novel’ toxin, called Am IT from the venom of scorpion Androctonus mauretanicus is isolated and characterized. A detailed analysis of the action of Am IT on insect axonal sodium currents is reported. Am IT was purified through gel filtration followed by C18 reversed-phase HPLC. Toxicity of Am IT in vivo was assessed on male German cockroach (Blattella germanica) larvae and C57/BL6 mice. Cross-reactivity of Am IT with two β-toxins was evidenced using 125I-iodinated toxin-based radioimmunoassays with synaptosomal preparations from rat brain. The complete amino acid sequence of Am IT was finally determined by Edman sequencing. Am IT was observed to compete with AaH IT4 purified from the venom of scorpion Androctonus australis in binding assays. It was recognized by an antibody raised against a β-type toxin, which indicated some structural similarity with β-toxins (or related toxin family). The ‘novel’ toxin exhibited dual activity since it competed with anti-mammal toxins in binding assays as well as showed contracting activity to insect. The toxin competed with radio-labeled β-toxin Css IV by binding to Na+ channels of rat brain synaptosomes. Analysis of toxin amino acid sequences showed that Am IT shares high structural identity (92%) with AaH IT4. In conclusion, Am IT not only reveals an anti-insect compound properties secreted by ‘Old World’ scorpions, paralyzing insect larvae by binding to Na+ channels on larvae’s nerve-cell membranes, but also exerts toxic activity in mice, which is similar to anti-mammal toxins from ‘New World’ scorpions (North and South Americas). Therefore, Am IT appears to be structurally and functionally similar to AaH IT4.

Key words: Androctonus mauretanicus venom; scorpion toxin; Am IT; β-toxin; anti-mammal toxin; anti-insect toxin; sodium channel; toxicity; de novo sequencing; Edman degradation

收稿日期：2015-01-06　　录用日期：2015-05-04

通讯作者：Naoual Oukkache　　E-mail: oukkache.naoual@gmail.com

引用本文：

Naoual Oukkache, Rachid ElJaoudi, Fatima Chgoury, Marisa Teixeira Rocha, Jean-Marc Sabatier. 一种源自北非蝎的抗昆虫毒素Am IT的结构与功能鉴定[J]. 生理学报 2015; 67 (3): 295-304.

Naoual Oukkache, Rachid ElJaoudi, Fatima Chgoury, Marisa Teixeira Rocha, Jean-Marc Sabatier. Characterization of Am IT, an anti-insect β-toxin isolated from the venom of scorpion Androctonus mauretanicus. Acta Physiol Sin 2015; 67 (3): 295-304 (in Chinese with English abstract).