芋螺多肽conantokins构效关系研究及中国南海织锦芋螺毒素的分离鉴定
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摘要
芋螺毒素由芋螺毒液管和毒囊内壁的毒腺分泌,多数由12~40个氨基酸组成,富含二硫键。与其他天然肽类毒素相比,芋螺毒素具有分子量小、结构稳定、高活性、高选择性及易于合成等突出优点。它们能特异性作用于乙酰胆碱受体及其它神经递质的各种受体亚型,及钙、钠、钾等多种离子通道,不仅可以直接作为药物,还可作为理想的分子模板用于发展新药先导化合物,对研究神经生物学也具有重要意义。本论文旨在研究conantokins及其类似物的镇痛活性,分离中国南海芋螺毒素,以及研究含一对及三对二硫键的芋螺多肽的合成及氧化折叠。
在conantokins及其类似物的合成、纯化及镇痛实验研究中:(1)合成了用于合成conantokins及其类似物的Fomc-Gla(tBu)2-COOH 10 g纯品;(2)在conantokins的合成中,针对某些难以耦合的氨基酸部位采用延长耦合时间来提高反应产率,合成了con-G、con-T、con-Qu及10个conantokins类似物;(3)采用凝胶层析、离子交换及RP-HPLC相结合的纯化方式对合成的conantokins进行了纯化,纯度均达到85%以上;(4)测定了conantokins及其突变体的镇痛活性,结果表明con-G的类似物con-G[S16Y]的镇痛活性显著高于其他conantokins及类似物,且镇痛活性与conantokins及类似物的NR2B亚基选择性有关。
系统分离鉴定了中国南海织锦芋螺的毒素组分,共获得了11个芋螺毒素,其中4个为新芋螺毒素,7个与已报道的相同。这些已知的毒素序列主要由Olivera B M等人通过cDNA克隆或分离获得,采用的织锦芋螺采自菲律宾宿务岛(Cebu)、马尼拉岛(Marinduquc)以及越南(Vietnam)周边地区,这些地区均濒临我国南海。通过序列对比及毒素成分对比,我们所采集的织锦芋螺品种与Olivera B M所采集的品种相近。分析比较主要织锦芋螺毒素的蛋白序列特征,观察到中国南海织锦芋螺中含有丰度很高的M族芋螺毒素,且序列较短。
为进一步研究芋螺毒素的结构及其他功能,我们又合成了含一对及三对二硫键的芋螺多肽,对其氧化折叠条件进行了研究。采用DMSO氧化体系合成了含一对二硫键的conantokins衍生物,采用不同助氧化还原系统,如L-半胱氨酸(Cys)、氧化型/还原型谷胱甘肽(GSSG/GSH)、二巯基苏糖醇(DTT)等,合成了含有三对二硫键的N-钙通道抑制剂SO-3的突变体SO-3[A3S,A4K,A12M],并对折叠的其他条件(如离子强度,温度等)进行了优化。
Conotoxins, which excreted from the venom gland of the venom canal, are mostly small, disulfide-rich peptides with 12~40 amino acids in length. Compared to other nature peptide toxins, conotoxins possess the superior chemical and pharmacological features, such as small molecular, stable structure, high activity and selectivity. Conotoxins can specifically target nicotinic acetylcholine receptors (AchRs), ion-channels and their subunits, such as calcium, sodium or potassium channel, and could be used as probes for the research of ion channel and receptor as well as new drugs or lead compounds. The purpose of the present paper was to probe the analgesic properties of conantokin peptides, isolate the conotoxins from Conus textile and probe the synthetic method of the conotoxins with one or three disulfide bonds.
In the studies of analgesic activity of conantokin peptides, the following results were obtained: (1) 10 g ofγ,γ’-Di-butyl-Nα-9-fluorenylmethyloxycarbonyl-γ- carboxyglutamic acid for conantokin peptide synthesis was synthesized; (2) Con-G, con-T, con-Qu and 10 conantokin analogues were synthesized with modified methods, such as longer coupling time for the special amino acids; (3) Conantokins and variants were purified by gel filtration chromatography, ion exchange chromatography as well as reversed phase liquid chromatography (RP-HPLC) with 85% or more purity; (4) Analgesic activities of the 13 conantokins and variants were evaluated by several pain models. The results showed that conantokins with higher NR2B subunit selectivity exhibited higher analgesic activity, con-G[S16Y] had the highest analgesic ability compared to the nature peptide con-G and other conantokin peptides.
The toxins of Conus textile from the South China Sea were isolated and identified. 11 conotoxins were obtained, 4 of them were first discovered, 7 of them had the similar amino acid sequences that come from Conus textile of Cebu and Marinduquc islands in Philippines or around areas of Vietnam Sea as reported by Olivera B M. The similar compositions of conotoxins of two specimen of the Conus textile suggest that they belong to same family. The special feature of conotoxins of Conus textile is the high content of M-family conotoxin with short amino acid sequence.
To further study the conotoxins structure and other biological activities, other conotoxins with 1 or 3 disulfide bonds were synthesized. Conantokin variants with one disulfide bond were synthesized using DMSO oxidation system, N-calcium ion
在conantokins及其类似物的合成、纯化及镇痛实验研究中:(1)合成了用于合成conantokins及其类似物的Fomc-Gla(tBu)2-COOH 10 g纯品;(2)在conantokins的合成中,针对某些难以耦合的氨基酸部位采用延长耦合时间来提高反应产率,合成了con-G、con-T、con-Qu及10个conantokins类似物;(3)采用凝胶层析、离子交换及RP-HPLC相结合的纯化方式对合成的conantokins进行了纯化,纯度均达到85%以上;(4)测定了conantokins及其突变体的镇痛活性,结果表明con-G的类似物con-G[S16Y]的镇痛活性显著高于其他conantokins及类似物,且镇痛活性与conantokins及类似物的NR2B亚基选择性有关。
系统分离鉴定了中国南海织锦芋螺的毒素组分,共获得了11个芋螺毒素,其中4个为新芋螺毒素,7个与已报道的相同。这些已知的毒素序列主要由Olivera B M等人通过cDNA克隆或分离获得,采用的织锦芋螺采自菲律宾宿务岛(Cebu)、马尼拉岛(Marinduquc)以及越南(Vietnam)周边地区,这些地区均濒临我国南海。通过序列对比及毒素成分对比,我们所采集的织锦芋螺品种与Olivera B M所采集的品种相近。分析比较主要织锦芋螺毒素的蛋白序列特征,观察到中国南海织锦芋螺中含有丰度很高的M族芋螺毒素,且序列较短。
为进一步研究芋螺毒素的结构及其他功能,我们又合成了含一对及三对二硫键的芋螺多肽,对其氧化折叠条件进行了研究。采用DMSO氧化体系合成了含一对二硫键的conantokins衍生物,采用不同助氧化还原系统,如L-半胱氨酸(Cys)、氧化型/还原型谷胱甘肽(GSSG/GSH)、二巯基苏糖醇(DTT)等,合成了含有三对二硫键的N-钙通道抑制剂SO-3的突变体SO-3[A3S,A4K,A12M],并对折叠的其他条件(如离子强度,温度等)进行了优化。
Conotoxins, which excreted from the venom gland of the venom canal, are mostly small, disulfide-rich peptides with 12~40 amino acids in length. Compared to other nature peptide toxins, conotoxins possess the superior chemical and pharmacological features, such as small molecular, stable structure, high activity and selectivity. Conotoxins can specifically target nicotinic acetylcholine receptors (AchRs), ion-channels and their subunits, such as calcium, sodium or potassium channel, and could be used as probes for the research of ion channel and receptor as well as new drugs or lead compounds. The purpose of the present paper was to probe the analgesic properties of conantokin peptides, isolate the conotoxins from Conus textile and probe the synthetic method of the conotoxins with one or three disulfide bonds.
In the studies of analgesic activity of conantokin peptides, the following results were obtained: (1) 10 g ofγ,γ’-Di-butyl-Nα-9-fluorenylmethyloxycarbonyl-γ- carboxyglutamic acid for conantokin peptide synthesis was synthesized; (2) Con-G, con-T, con-Qu and 10 conantokin analogues were synthesized with modified methods, such as longer coupling time for the special amino acids; (3) Conantokins and variants were purified by gel filtration chromatography, ion exchange chromatography as well as reversed phase liquid chromatography (RP-HPLC) with 85% or more purity; (4) Analgesic activities of the 13 conantokins and variants were evaluated by several pain models. The results showed that conantokins with higher NR2B subunit selectivity exhibited higher analgesic activity, con-G[S16Y] had the highest analgesic ability compared to the nature peptide con-G and other conantokin peptides.
The toxins of Conus textile from the South China Sea were isolated and identified. 11 conotoxins were obtained, 4 of them were first discovered, 7 of them had the similar amino acid sequences that come from Conus textile of Cebu and Marinduquc islands in Philippines or around areas of Vietnam Sea as reported by Olivera B M. The similar compositions of conotoxins of two specimen of the Conus textile suggest that they belong to same family. The special feature of conotoxins of Conus textile is the high content of M-family conotoxin with short amino acid sequence.
To further study the conotoxins structure and other biological activities, other conotoxins with 1 or 3 disulfide bonds were synthesized. Conantokin variants with one disulfide bond were synthesized using DMSO oxidation system, N-calcium ion
引文
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[2]Terlau H, Olivera B M. Conus Venoms: a rich source of novel ion channel-targeted peptides[J]. Physiol Rev, 2004, 84(1): 41-68.
[3]岳俊杰,戴秋云,黄培堂,王国力,梁龙,俞炜源,黄培堂. 芋螺毒素与钙通道相互作用的计算计模拟[J]. 生物技术通讯, 2002, 13(4): 281-285.
[4]McIntosh J M, Olivera B M, Cruz L J, Corpuz G P, Jones R M, Garrett J E. Conotoxin peptides[P]. US: 6,767,896,2004-7-27.
[5]Olivera B M, Cruzab L J. Conotoxins, in retrospect[J]. Toxincon, 2001, 39(1): 7-14.
[6]Layer R T, Wagstaff J D, White H S. Conantokins: Peptide Antagonists of NMDA Receptors[J]. Curr Med Chem, 2004, 11(23): 3073-3084.
[7]McIntosh J M, Olivera B M, Cruz L J, Gray W R. γ-Carboxyglutamate in a neuroactive toxin[J]. J Biol Chem, 1984, 259(23): 14343-14346.
[8]Haack J A, Revier J, Parks T N, Mena E E, Cruz L J, Olivera B M. Conantokin-T. A γ-Carboxyglutamate-containing peptide with N-methyl-D-aspartate antagonist activity[J]. J Biol Chem, 1990, 265(11): 6025-6029.
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