中国南海三种芋螺毒素的分离及鉴定
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摘要
芋螺属腹足纲软体动物,全世界约有500～700种,遍布世界各暖海区,我国有芋螺100余种,主要分布在西沙群岛、海南岛及台湾海域。芋螺毒素由芋螺毒液管和毒囊内壁的毒腺所分泌,每种芋螺的毒液中含50～200个活性多肽,且每种芋螺的毒素序列极少重叠。芋螺毒素多数由10～40个氨基酸残基组成,富含二硫键,可作用于各种离子通道和受体的类型及亚型。它们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,对神经生物学也具有重要意义。本论文旨在研究发现及鉴定新的芋螺毒素种类,从织锦芋螺,堂皇芋螺和大理石芋螺中分离、纯化、鉴定新的芋螺多肽,为进一步研究它们的生理及药理功能奠定基础。
我们于2007年4月在我国南海地区采集了三种芋螺,进行芋螺毒素的分离、纯化和鉴定。
在芋螺毒素的初分离阶段,采用G-25凝胶层析、离子交换、RP-HPLC相结合的纯化方式对三种芋螺中的芋螺毒素进行分离纯化,共获得了53种芋螺毒素,其中包括23种织锦芋螺毒素,25种堂皇芋螺毒素和5种大理石芋螺毒素。对分离纯化的芋螺毒素进行了HPLC分析,证实了获得的芋螺毒素的纯度均达到85%以上。
在芋螺毒素的鉴定试验中,我们首先用基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)方法确定其分子量,它们的分子量均在1000到4000之间。采用了Edman降解及四极杆-飞行时间串联质谱两种测序方法,对新发现的芋螺毒素进行了序列分析,共得到17个新的芋螺毒素序列,包括8个织锦芋螺毒素序列、8个堂皇芋螺毒素和1个大理石芋螺毒素序列,并对它们的序列特点及可能存在的生物活性进行了初步分析。
Cone snails are predatory gastropod mollusks distributed in all tropical marine habitat with 500~700 species all over the world. There are about 100 conus species in China, mostly distributed in Xisha, Hainan islands and Taiwan water areas.
Conotoxins are secreted by venom duct and theca. Conus venom has 50～200 different bioactive peptides with little overlap between species. Generally, conotoxins contain 10~40 amino acid residues with rich disulfide bond and could target a series of ion channels or recepter subtypes.Conotoxins are not only developed directly into drugs, but also used as perfect drug design templates and probes for neurobiological researches. This research is to isolate, purify and identify new conotoxins from conus textile, conus imperialis and conus marmoreus in South China Sea and lays a foundation for further researches on their physiological and pharmacological functions.
The living specimens of the three conus for the isolation, purification and identification were collected from the South China Sea in Apr,2007.
A series of isolation methods, such as Sephadex G-25,ion exchange and RP-HPLC were used to isolate and purify the conotoxins from the above three cone snails. 53 conotoxins were isolated, including 23, 25 and 5 peptides from conus textile, conus imperialis and conus marmoreus, respectively. The purity reaches more than 85 percent by the analysis of HPLC.
The molecular weights were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The new peptide sequences were analyzed by automatic Edman sequencr and Quatropde-Time of Flight Mass spectrometer. A total of 17 new peptides, including 8,8 and 1 novel peptides were found for the first time in conus textile, conus imperialis and conus marmoreus, respectively. The related biological activities were predicted based on their sequence features.
我们于2007年4月在我国南海地区采集了三种芋螺,进行芋螺毒素的分离、纯化和鉴定。
在芋螺毒素的初分离阶段,采用G-25凝胶层析、离子交换、RP-HPLC相结合的纯化方式对三种芋螺中的芋螺毒素进行分离纯化,共获得了53种芋螺毒素,其中包括23种织锦芋螺毒素,25种堂皇芋螺毒素和5种大理石芋螺毒素。对分离纯化的芋螺毒素进行了HPLC分析,证实了获得的芋螺毒素的纯度均达到85%以上。
在芋螺毒素的鉴定试验中,我们首先用基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)方法确定其分子量,它们的分子量均在1000到4000之间。采用了Edman降解及四极杆-飞行时间串联质谱两种测序方法,对新发现的芋螺毒素进行了序列分析,共得到17个新的芋螺毒素序列,包括8个织锦芋螺毒素序列、8个堂皇芋螺毒素和1个大理石芋螺毒素序列,并对它们的序列特点及可能存在的生物活性进行了初步分析。
Cone snails are predatory gastropod mollusks distributed in all tropical marine habitat with 500~700 species all over the world. There are about 100 conus species in China, mostly distributed in Xisha, Hainan islands and Taiwan water areas.
Conotoxins are secreted by venom duct and theca. Conus venom has 50～200 different bioactive peptides with little overlap between species. Generally, conotoxins contain 10~40 amino acid residues with rich disulfide bond and could target a series of ion channels or recepter subtypes.Conotoxins are not only developed directly into drugs, but also used as perfect drug design templates and probes for neurobiological researches. This research is to isolate, purify and identify new conotoxins from conus textile, conus imperialis and conus marmoreus in South China Sea and lays a foundation for further researches on their physiological and pharmacological functions.
The living specimens of the three conus for the isolation, purification and identification were collected from the South China Sea in Apr,2007.
A series of isolation methods, such as Sephadex G-25,ion exchange and RP-HPLC were used to isolate and purify the conotoxins from the above three cone snails. 53 conotoxins were isolated, including 23, 25 and 5 peptides from conus textile, conus imperialis and conus marmoreus, respectively. The purity reaches more than 85 percent by the analysis of HPLC.
The molecular weights were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The new peptide sequences were analyzed by automatic Edman sequencr and Quatropde-Time of Flight Mass spectrometer. A total of 17 new peptides, including 8,8 and 1 novel peptides were found for the first time in conus textile, conus imperialis and conus marmoreus, respectively. The related biological activities were predicted based on their sequence features.
引文
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[3]Mondal S, Babu R M, Bhavna R. I-conotoxin superfamily revisited. J Pept Sci, 2006, 12: 679 ~85.
[4]Norton R S, Olivera B M. Conotoxins down under. Toxicon,2006,48:780~98.
[5]Terlau H, Oliver B M. Conus Venoms: a rich source of novel ion channel– targeted peptides.Physiol Rev, 2004, 84:41~68.
[6]Woodward S R, Cruz L J, Olivera B M. Constant and hypervariable regions in conotoxins propeptides. EMBOJ, 1990, 9(4):1015~1020.
[7]Conticello S G, Gilad Y, Avidan N. Machanisms for evolving hypervariability: the case of conopeptides. Mol Biol Evol, 2001, 18 (2):120~131.
[8]Kauferstein S, Melaun C, Mebs D. Direct Cdna cloning of novel conopeptide precursor of the O-superfamily. Peptides, 2005, 26 (3):361~367.
[9]张伟,韩禹宏. O-超家族芋螺毒素研究进展.生命化学,2005,17(5):404~410.
[10]王承忠,蒋辉,戚正武.芋螺毒素研究进展.生物化学与生物物理进展,2003,30(4):537~545.
[11]Olivera B M, Cruzab L J . Conotoxins, in retrospect . Toxicon , 2001, 39 (1) : 7~14.
[12]Jimenez E C, Watkins M, Juszczak L J. Contryphans from Conus textile venom ducts. Toxicon, 2001, 39:803~808.
[13]Jacobsen R, Jimenez E C, De la Cruz RGC. A novel D- leucine- containing Conus peptide: diverse confomational dynamics in the contryphan family. J. Peptide Res, 1999, 54:93~99.
[14]Lewis R J. Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters. IUBMB Life, 2004, 56(2):89~93.
[15]Livett B G, Gayler K R, Khalil Z. Drugs from the sea:conopeptides as potential therapeutics. Curr Med Chem, 2004, 11(13):1715~1723.
[16]Terlau H, Olivera B M. Conus venoms:a rich source of novel ion channel-targeted peptides. Physiol Rev, 2004, 84(1):41~68.
[17]吴雪尘,彭灿.芋螺毒素国内外研究现状,科技资讯,2007,10:209~210.
[18]陈冀胜.药物开发的新领域.中国天然药物,2004,(2):129~134.
[19]应士波,严小军.芋螺毒素的药用价值研究进展.天然产物研究与开发,2006,18:138~143.
[20]长孙东亭,罗素兰,误勇.海南产大蔚芋螺的新型O-超家族芋螺毒素基因克隆.中国海洋药物杂志,2007,26(1):1~7.
[21]Alonso D, Khalilb Z, Livettc B G. Drugs from the sea :conotoxins as drug leads for neuropathic pain and other neurological conditions. Mini Reviews in Medicinal Chemistry, 2003, 3 :785~787.
[22]Armishaw C J, Alewood P F. Conotoxins as Research Tools and Drug Leads.Current Protein and Peptide Science, 2005 (6):221~225.
[23]Santos A D, McIntosh J M, Hillyard DR.The A-superfamily of conotoxins, The journal of biological Chemistry,2004,279,(17):17596~17606.
[24]Luo S, Kulak J M, Cartier G E. Alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. J Neurosci, 1998, 18(21):8571~8579.
[25]Kauferstein S, Huys I, Kuch U. Novel conopeptides of the I-superfamily occur in several clades of cone snails.Toxicon, 2004, 44(5):539~548.
[26]Zugasti-Cruz A, Aguilar M B, Falco′n A. Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice. Peptides, 2008,29:179~185.
[27]Luo S L, Zhangsun D T,Zhang B. Direct cDNA cloning of novel conotoxins of the T-superfamily from conus textile.Peptides, 2006, 27:2640~2646.
[28]卢松柏,于芳,王建华.从织锦芋螺中克隆α芋螺毒素序列,中国生物化学与分子生物学报,1999,15(3):413~416.
[29]Tavazoie S F, Tavazoie M F, Mcintosh J M. Differential block of nico- tinic synapses on B versus C neurones in sympathetic ganglia of frog by alpha-cono- Toxins M II and M I B. J Pharmacol, 1997, 120 (6) : 995～1000.
[30]Olivera B M,Cartier G E, Watkins M. O-superfamily conotoxin peptides[P].US:6,762,165, 2004 -7-13.
[31]Corpuz G P, Jacobsen R B, Jimenez EC. Definition of the M-conotoxin superfamily:characterization of novel peptides from molluscivorous Conus venoms. Biochemistry, 2005, 44(22):8176~8186.
[32]Garrett J E, Buczek O, Watkins M. Biochemical and gene expression analyses of conotoxins in Conus textile venom ducts. Biochem Biophys Res Commun, 2005, 28(1): 362~367.
[33]Hillyard D R, Olivera B M, Woodward S R. A molluscivorous conus toxin: conserved frameworks in conotoxins. Biochemistry, 1989, 28:358~361.
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