中国南海新芋螺多肽的合成及结构改造
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摘要
芋螺毒素是近二十年来国际上的研究热点。芋螺毒素由芋螺毒液和毒囊内壁的毒腺所分泌,多数由12-40个氨基酸组成,富含二硫键,能特异作用于乙酰胆碱受体及其它神经递质的各种受体亚型,以及钙、钠、钾等多种离子通道。世界上芋螺资源丰富,在我国主要分布在西沙群岛、南海及台湾海域。本实验室在中国南海芋螺的采集、毒素分离、纯化、基因克隆、合成及活性测定等方面进行了系列工作。本论文在已有工作的基础上,开展如下三方面研究:(1)ω-芋螺毒素SO-3的结构与活性关系;(2)中国南海α-家族芋螺毒素的合成及结构改造;(3)中国南海几种新的芋螺毒素的合成。
在SO-3的结构与活性关系研究中:设计合成了7个SO-3和MⅦA嵌合体,测定了其金鱼毒性及镇痛活性,发现影响SO-3毒性较低的关键氨基酸处于1及2,2的氨基酸影响更显著。
在中国南海α-家族芋螺毒素的合成及结构改造方面:(1)合成了7个α-家族芋螺毒素,并对它的镇痛活性进行评价,发现4个多肽具有显著的镇痛活性。(2)设计合成了1个双功能小分子多肽。在GI分子的N-端引入小分子钠通道抑制剂后,新分子具有部分钠通道抑制活性,对金鱼的毒性显著减低,其对烟碱型乙酰胆碱受体的作用正在测定中。(3)设计合成了2个Vc1.1和GI突变物。(4)利用α-芋螺毒素的骨架(CC-C-C)及天然堂皇芋螺毒素的氨基酸组成,设计合成了一个由11个氨基酸组成的线性短肽,确定了二硫键连接方式,发现其有较高的镇痛活性。
在中国南海新克隆芋螺毒素的合成及镇痛活性研究中:(1)合成了6个T-超家族芋螺毒素,初步研究表明,这些毒素在较大剂量下对金鱼不致死,且部分具有兴奋作用。(2)合成了6个新克隆芋螺多肽,其中有4个肽由于疏水性较强而没有得到目标产物。在大鼠坐骨神经半切模型中,一个新的M型芋螺毒素L-6 (Mr6)具有强烈的镇痛活性且呈剂量依赖性。
上述研究为确定ω-芋螺毒素SO-3的毒性、活性位点,发现具有镇痛应用前景的新型芋螺多肽,作出了新贡献。
Conotoxins have been intensively studied during last twenty years. Conotoxin excreted from the venom gland of the venom canal are mostly small, disulfide-rich peptides with 12～40 amino acids in length, and specifically target nicotinic acetylcholine receptors, ion-channels and their subunits, such as calcium, sodium and potassium ion channel. Conus resources exist widely in the world, specifically, are mainly distributed in the Paracel Islands, South China Sea and the waters around Taiwan. A series of studies on conotoxins including: the collection of conus, toxin isolation, purification, gene cloning, synthesis and activity assay, have been carried on in our laboratory. The thesis mainly focused on the three aspects in conotoxins: (1) The structure-activity relationship of theω-conotoxins SO-3; (2) Syntheses and modifications ofα-conotoxins from South China Sea; (3) Syntheses of several new conotoxins from South China Sea.
In the studies of the structure-activity relationship of theω-conotoxins SO-3, seven variants of SO-3 and MⅦA were designed and synthesized, toxicity and analgesic activity were evaluated, and the effect of key amino acids in 1 and 2 on the toxicity of SO-3 were discovered.
In the studies of syntheses and modifications ofα-conotoxins from South China sea: (1) Sevenα-conotoxins were synthesized and evaluated for their analgesic activity, and four peptides of them were found to have significant analgesic activity. (2) A low molecular difunctional peptide, which a small-molecule sodium channel inhibitor is attached in the N-terminal of GI, was designed and synthesized and showed inhibitory activity in sodium channels and low toxicity to the goldfish compared with GI. (3) Two variants of Vc1.1 and GI were designed and synthesized. (4) A small peptide of 11 amino acids has been designed and synthesized based on cysteine framework (CC-C-C) ofα-conotoxins disulfide connectivity and exhibited high analgesic activity.
In the studies of several novel conotoxins from South China Sea: (1) Six T-superfamily conotoxins were synthesized and their toxicity were determined with goldfish. The results indicated that these T-superfamily conotoxins have no toxicity and some of them show exciting activity. (2) Six novel conotoxin linear peptides were synthesized but four final products were not obtained because of the difficulty in folding. The analgesic activity of a M-superfamily conotoxin L-6 (Mr6) was determined with PNL model in rats. The results indicated that Mr6 has potent analgesic activity in dose-dependent mode.
The above studies will contribute to determination ofω-conotoxins SO-3 toxicity and active site, and discovery of novel analgesic conus peptides.
在SO-3的结构与活性关系研究中:设计合成了7个SO-3和MⅦA嵌合体,测定了其金鱼毒性及镇痛活性,发现影响SO-3毒性较低的关键氨基酸处于1及2,2的氨基酸影响更显著。
在中国南海α-家族芋螺毒素的合成及结构改造方面:(1)合成了7个α-家族芋螺毒素,并对它的镇痛活性进行评价,发现4个多肽具有显著的镇痛活性。(2)设计合成了1个双功能小分子多肽。在GI分子的N-端引入小分子钠通道抑制剂后,新分子具有部分钠通道抑制活性,对金鱼的毒性显著减低,其对烟碱型乙酰胆碱受体的作用正在测定中。(3)设计合成了2个Vc1.1和GI突变物。(4)利用α-芋螺毒素的骨架(CC-C-C)及天然堂皇芋螺毒素的氨基酸组成,设计合成了一个由11个氨基酸组成的线性短肽,确定了二硫键连接方式,发现其有较高的镇痛活性。
在中国南海新克隆芋螺毒素的合成及镇痛活性研究中:(1)合成了6个T-超家族芋螺毒素,初步研究表明,这些毒素在较大剂量下对金鱼不致死,且部分具有兴奋作用。(2)合成了6个新克隆芋螺多肽,其中有4个肽由于疏水性较强而没有得到目标产物。在大鼠坐骨神经半切模型中,一个新的M型芋螺毒素L-6 (Mr6)具有强烈的镇痛活性且呈剂量依赖性。
上述研究为确定ω-芋螺毒素SO-3的毒性、活性位点,发现具有镇痛应用前景的新型芋螺多肽,作出了新贡献。
Conotoxins have been intensively studied during last twenty years. Conotoxin excreted from the venom gland of the venom canal are mostly small, disulfide-rich peptides with 12～40 amino acids in length, and specifically target nicotinic acetylcholine receptors, ion-channels and their subunits, such as calcium, sodium and potassium ion channel. Conus resources exist widely in the world, specifically, are mainly distributed in the Paracel Islands, South China Sea and the waters around Taiwan. A series of studies on conotoxins including: the collection of conus, toxin isolation, purification, gene cloning, synthesis and activity assay, have been carried on in our laboratory. The thesis mainly focused on the three aspects in conotoxins: (1) The structure-activity relationship of theω-conotoxins SO-3; (2) Syntheses and modifications ofα-conotoxins from South China Sea; (3) Syntheses of several new conotoxins from South China Sea.
In the studies of the structure-activity relationship of theω-conotoxins SO-3, seven variants of SO-3 and MⅦA were designed and synthesized, toxicity and analgesic activity were evaluated, and the effect of key amino acids in 1 and 2 on the toxicity of SO-3 were discovered.
In the studies of syntheses and modifications ofα-conotoxins from South China sea: (1) Sevenα-conotoxins were synthesized and evaluated for their analgesic activity, and four peptides of them were found to have significant analgesic activity. (2) A low molecular difunctional peptide, which a small-molecule sodium channel inhibitor is attached in the N-terminal of GI, was designed and synthesized and showed inhibitory activity in sodium channels and low toxicity to the goldfish compared with GI. (3) Two variants of Vc1.1 and GI were designed and synthesized. (4) A small peptide of 11 amino acids has been designed and synthesized based on cysteine framework (CC-C-C) ofα-conotoxins disulfide connectivity and exhibited high analgesic activity.
In the studies of several novel conotoxins from South China Sea: (1) Six T-superfamily conotoxins were synthesized and their toxicity were determined with goldfish. The results indicated that these T-superfamily conotoxins have no toxicity and some of them show exciting activity. (2) Six novel conotoxin linear peptides were synthesized but four final products were not obtained because of the difficulty in folding. The analgesic activity of a M-superfamily conotoxin L-6 (Mr6) was determined with PNL model in rats. The results indicated that Mr6 has potent analgesic activity in dose-dependent mode.
The above studies will contribute to determination ofω-conotoxins SO-3 toxicity and active site, and discovery of novel analgesic conus peptides.
引文
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[2] Buczek O, Bulaj G, Olivera B M. Conotoxins and the posttranslational modification of secreted gene products [J]. Cellular and Molecular Sciences, 2005, 62(24):3067-3079.
[3] Olivera B M, Cruzab L J. Conotoxins in retrospect [J]. Toxicon, 2001, 39(1):7-14.
[4] Shon K, Olivera B M, Watkins M, et al.μ-conotoxin PⅢA, a new peptide for discrimination among tetrodotoxin-sensitive Na channel subtypes [J]. Neuroscience, 1998, 18:4473-4481.
[5] Walker C S., Steel D, Jacobsen R B, et al. The T-superfamily of conotoxins [J]. Biological Chemistry, 1999, 274(43):30664-30671.
[6] Zamponi G W, Lewis R J, Todorovic S M., Stephen P. Arneric, Terrance P. Snutch. Role of voltage-gated calcium channels in ascending pain pathways [J]. Brain Research Reviews, 2009, 60:84-89.
[7] Motin L, Adams D J.ω-Conotoxins inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn [J]. Neuropharmacology, 2008, 55:860-864.
[8] Bingham J, Mitsumaga E, Bergeron Z L. Drgus from slugs-Past, present and future perspectives ofω-conotoxin research [J]. Chemico-Biological Interactions, 2010, 183:1-18.
[9] Sxhulz J R, Norton A G, Gilly W F, et al.The projectile tooth of a fish-hunting cone snail: Conus catus injects venom into fish prey using a high-speed ballistic mechanism [J]. Biological Bulletin, 2004, 207(2): 77-79.
[10] Kohn A J. Piscivorous gastropods of the genus Conus [J]. National Academy of the Sciences, 1956, 42(3):168-171
[11] Zhan Li, He XiangPing, Xie ZuoPing, et al. Effect of new o-superfamily contoxin SO-3 on sodium and potassium currents of cultured hippocampal neurons [J]. Brain Research, 2003 965 (1-2):155-158.
[12] Basus V J, Nadasdi L, Ramachandran J,et al.Solution structure ofω-conotoxin MⅦA using 2D NMR spectroscopy [J].FEBS, 1995, 370(3):163-167.
[13]闫永彬,涂光忠,罗雪春,等.ω-芋螺毒素SO-3溶液构象的核磁共振研究[J].科学通报, 2003, 48(7):677
[14] Kim J I, Takahashi M, Martin-Moutot N, et al. Tyr13 is essential for the binding ofω–conotoxin MⅦC to the P/Q-type calcium channel [J]. Biochemical and Biophysical Communications, 1995, 214:305-309.
[15] Nadasdi L, Yamashiro D, Chung D, et al. Structure activity analysis of a conus peptide blocker of N-type neuronanl calcium channels [J]. Biochemistry, 1995, 34:8076-8081.
[16] Kim J I, Takahashi M, Ogura A, et al. Hydroxyl group of Tyr13 is essential for the activity ofω-conotoxin GⅥA, a peptide toxin for N-type calcium channel [J]. Biological Chemistry,
1994, 269:23876-23878.
[17] Sato K, Park N G, Kohno T, et al. Role of basic residues for the binding ofω-conotoxin GⅥA to N-type calcium channels [J]. Biochemical and Biophysical Communications, 1993, 194:1292-1296.
[18] Kim J I, Takahashi M, Martin-Moutor N, et al. Tyr13 is essential for the binding ofω-conotoxin MⅥC to the P/Q-type calcium channel [J]. Biochemical and Biophysical Communications, 1995, 214:305-309.
[19]戴秋云,刘凤云,周艳荣,等.ω-芋螺毒素MⅦC的N及C端修饰对折叠及活性影响[J].中国生物化学与分子生物学报, 2001, 17(1):66-70.
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