海南敬钊缨毛蛛肽类毒素与虎纹捕鸟蛛非肽类毒素的结构与功能研究
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摘要
蜘蛛毒素在药理学、神经生物学和医学领域已展现出广阔的应用前景，对蜘蛛毒素的研究具有重要的意义。蜘蛛毒素中神经毒素是最重要的。
海南敬钊缨毛蛛(Chilobrachys jingzhao)毒素是最近在海南发现的蜘蛛新种，该蜘蛛粗毒中含有多种活性多肽和蛋白质成分。通过离子交换和反相HPLC分离纯化出两种多肽类毒素分别命名为海南敬钊缨毛蛛毒素-Ⅱ(Jingzhao-Ⅱ，JZTX-Ⅱ)和海南敬钊缨毛蛛毒素-Ⅲ(Jingzhao-Ⅲ，JZTX-Ⅲ)。JZTX-Ⅱ和JZTX-Ⅲ的氨基酸序列全部测出，分别由34和36个氨基酸组成，含有6个半胱氨酸，形成3对二硫键。MALDI-TOF质谱测定它们的相对分子质量分别为3726.35Da和3918.46Da。JZTX-Ⅱ对电刺激诱导的小鼠膈神经-膈肌收缩产生抑制作用，浓度为1.0×10~(-6)mol/L时的阻断时间为15分钟左右，对钠通道和延迟调整型钾通道的激活和失活没有影响。通过同源性搜索，它与蜘蛛Hysterocrates gigas中的SNX-482毒素具有63％的同源性，而SNX-482能阻断人的E系钙通道，该毒素对其他K~+、Na~+、Cl~-、Ba~(2+)及其它类型的Ca~(2+)离子通道都没有作用。根据JZTX-Ⅱ与毒素SNX-482高度的同源性，可推测JZTX-Ⅱ很可能也是一种E型钙通道阻断剂。JZTX-Ⅲ对电刺激诱导的小鼠膈神经-膈肌的收缩没有作用，小鼠脑室注射5分钟致死，能阻断表达在非洲爪蟾(Xenopus Laevis)卵母细胞上的hH1(Nav1.5)有阻断作用，半有效浓度为144.5nmol／L。
虎纹捕鸟蛛(Ornithoctonus huwena)是生活在我国南部云南、广西的山区，本实验室在其粗毒中发现了许多神经肽类毒素，但对其中的非肽类组分研究甚少。采用两次反相高效液相色谱从虎纹捕鸟蛛中分离出一种相对分子质量为147Da的成分，命名为HWTXs-Ⅰ。经质谱、红外光谱、核磁共振等方法鉴定为一类酰胺类物质，该毒素可以阻断蟾蜍离体灌流心脏的跳动，其功能与乙酰胆碱相似但结构相差很大。将该小分子成分与标准的谷氨酰胺、乙酰胆碱红外光
.1.义摘’丝
潜、核磁共振进行对照，三者差别很人。据此我们推断该毒素分子
可能为一种拟胆碱类的新成分。根据核磁共振谱推测其基本结构单
元有两个亚甲基和一个次甲基。
The spider Chilobrachys jingzhao was recently identified as a new specice. The crude venom contains a mixture of compounds with different types of biological activities. Two novel Peptide Neueotoxins were purified from the venom of Chilobrachys jingzhao by using combination of ion-exchange and reverse phase HPLC. The complete amino acid sequences of them were obtained by automated Edman degradation,and the biological functions of them have been investigated on several experimental models.
They are named as JZTX-Ⅱ and JZTX-Ⅲ, respectively. The masses of the two toxins are determined by the MALDTI-TOF MS as 3726. 35Da
and 3918. 46Da, respectively.The sequences of the two toxins are determined and found to consist of 34 and 36 amino acid residues, respectively.Both of the two sequences include six Cys residues, which form three disulphide bonds. JZTX-Ⅱ can block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm . JZTX- Ⅱ show 63% identity with SNX-482 toxin,a neurotoxin isolated from the spider Hysterocrates gigas. Based on high sequence identity with SNX-482, it was estimated that JZTX-Ⅱ could also block the E-type calcium channel just like SNX-482, which is accepted as a blocker of E-type calcium channel. JZTX-Ⅲ could block the sodium channel hHl(Navl.5) which was expressioned in the Xenopus occytes.
The Chinese bird spider Ornithoctonus huwena is distributed in the hilly areas of Yunan and Guangxi in the south of China. So far several neurotoxins have been isolated from the venom of spider Ornithoctonus huwena. A novel low molecular compound which was effective to heart is isolated from the venom of the spider through two times reverse phase High performance liquid chromatography.Its exact molecular weight is 147Da determined by
ESI mass spectrum. According to its MS, NMR, IR, the toxin has been proved as apoloyamine amides. The compound showed effects on isolated atria, although the structure of this compound is different from the Ach, the bioactivities of them are similar to each other.
海南敬钊缨毛蛛(Chilobrachys jingzhao)毒素是最近在海南发现的蜘蛛新种，该蜘蛛粗毒中含有多种活性多肽和蛋白质成分。通过离子交换和反相HPLC分离纯化出两种多肽类毒素分别命名为海南敬钊缨毛蛛毒素-Ⅱ(Jingzhao-Ⅱ，JZTX-Ⅱ)和海南敬钊缨毛蛛毒素-Ⅲ(Jingzhao-Ⅲ，JZTX-Ⅲ)。JZTX-Ⅱ和JZTX-Ⅲ的氨基酸序列全部测出，分别由34和36个氨基酸组成，含有6个半胱氨酸，形成3对二硫键。MALDI-TOF质谱测定它们的相对分子质量分别为3726.35Da和3918.46Da。JZTX-Ⅱ对电刺激诱导的小鼠膈神经-膈肌收缩产生抑制作用，浓度为1.0×10~(-6)mol/L时的阻断时间为15分钟左右，对钠通道和延迟调整型钾通道的激活和失活没有影响。通过同源性搜索，它与蜘蛛Hysterocrates gigas中的SNX-482毒素具有63％的同源性，而SNX-482能阻断人的E系钙通道，该毒素对其他K~+、Na~+、Cl~-、Ba~(2+)及其它类型的Ca~(2+)离子通道都没有作用。根据JZTX-Ⅱ与毒素SNX-482高度的同源性，可推测JZTX-Ⅱ很可能也是一种E型钙通道阻断剂。JZTX-Ⅲ对电刺激诱导的小鼠膈神经-膈肌的收缩没有作用，小鼠脑室注射5分钟致死，能阻断表达在非洲爪蟾(Xenopus Laevis)卵母细胞上的hH1(Nav1.5)有阻断作用，半有效浓度为144.5nmol／L。
虎纹捕鸟蛛(Ornithoctonus huwena)是生活在我国南部云南、广西的山区，本实验室在其粗毒中发现了许多神经肽类毒素，但对其中的非肽类组分研究甚少。采用两次反相高效液相色谱从虎纹捕鸟蛛中分离出一种相对分子质量为147Da的成分，命名为HWTXs-Ⅰ。经质谱、红外光谱、核磁共振等方法鉴定为一类酰胺类物质，该毒素可以阻断蟾蜍离体灌流心脏的跳动，其功能与乙酰胆碱相似但结构相差很大。将该小分子成分与标准的谷氨酰胺、乙酰胆碱红外光
.1.义摘’丝
潜、核磁共振进行对照，三者差别很人。据此我们推断该毒素分子
可能为一种拟胆碱类的新成分。根据核磁共振谱推测其基本结构单
元有两个亚甲基和一个次甲基。
The spider Chilobrachys jingzhao was recently identified as a new specice. The crude venom contains a mixture of compounds with different types of biological activities. Two novel Peptide Neueotoxins were purified from the venom of Chilobrachys jingzhao by using combination of ion-exchange and reverse phase HPLC. The complete amino acid sequences of them were obtained by automated Edman degradation,and the biological functions of them have been investigated on several experimental models.
They are named as JZTX-Ⅱ and JZTX-Ⅲ, respectively. The masses of the two toxins are determined by the MALDTI-TOF MS as 3726. 35Da
and 3918. 46Da, respectively.The sequences of the two toxins are determined and found to consist of 34 and 36 amino acid residues, respectively.Both of the two sequences include six Cys residues, which form three disulphide bonds. JZTX-Ⅱ can block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm . JZTX- Ⅱ show 63% identity with SNX-482 toxin,a neurotoxin isolated from the spider Hysterocrates gigas. Based on high sequence identity with SNX-482, it was estimated that JZTX-Ⅱ could also block the E-type calcium channel just like SNX-482, which is accepted as a blocker of E-type calcium channel. JZTX-Ⅲ could block the sodium channel hHl(Navl.5) which was expressioned in the Xenopus occytes.
The Chinese bird spider Ornithoctonus huwena is distributed in the hilly areas of Yunan and Guangxi in the south of China. So far several neurotoxins have been isolated from the venom of spider Ornithoctonus huwena. A novel low molecular compound which was effective to heart is isolated from the venom of the spider through two times reverse phase High performance liquid chromatography.Its exact molecular weight is 147Da determined by
ESI mass spectrum. According to its MS, NMR, IR, the toxin has been proved as apoloyamine amides. The compound showed effects on isolated atria, although the structure of this compound is different from the Ach, the bioactivities of them are similar to each other.
引文
[1] Roderick H. Scott, KathyG. Sutton and Annette C. Dolphin. Trend in Neurosciences, 1993; 16(4): 153-159
[2] 陈远聪，生命的化学，1991；11(3)：1-2
[3] 王贤纯，博士毕业论文，1998
[4] Eugene. Grinshin, Eur. J. Biochem, 1999; 264: 276-280
[5] 梁宋平，潘欣。生物化学与生物物理进展，1994；21(5)：390—395
[6] Longenecker H E Tr, Hurlbut W P, Mauro A et al. Nature, 1970; 225: 701
[7] Frontail N, Ceccarelli B, Gorio A et al. J Cell Biol, 1976: 68: 462
[8] Hurlbut. W P., Iezzi. N, Fesce. R. &Ceccarelli. B, J. Physiol, 1990; 425: 501-526
[9] Petrenko A G, Kovalenko V A, Shamotienko Z N et al. EMBO Journal, 1990; 9: 2023
[10] Kiyatkin N I, Dulubova I E, Cheknovskaya I A et al. FEBS Letts, 1990; 270: 127
[11] Lurnev, A. V., Oemin. V. V. etal, Bioong. Khim, 1991; 171021-1026
[12] Erishin E V. Toxicon, 1998 Nov; 36(11): 1693-701
[13] Michaely. p. &Bennett. VTrends, Cell Biol, 1992; 2: 127-129
[14] Danilevich V N, Lukianov S A, Grishin E V. Bioorg Khim. 1999 Jul; 25(7): 537-547
[15] Sheumack D D, Classens R, Whiteleg N M et al. FEBS Letts, 1985; 181: 154
[16] Brown M R, Sheumack D D, Tyler M I et al. Biochem J, 1988; 250: 401
[17] Nicholson G M, Little M J, Tyler M, Narahashi T. Toxicon, 1996 Nov-Dec; 34(11-12): 1443-1453
[18] Skinner W S, Adams M E, Quistad et al. J, Biol Chem, 1989; 264: 2150
[19] Adams M E, Bindokas V P, Hasewage L&Veneva V J. J Biol Chem, 1990; 265: 861-867
[20] Ertel E A, Waren V A, Adams M E, Griffin P R etal. Bioc
hemistry, 1994; 33: 5098-5208
[21] Mintz I M, Venema V J, Adams M E&Bean B P. Proc. Natl. A cad. Sci. USA, 1991; 88: 6628-6631
[22] Mintz I M Venema V J, Swiderek K M et al. Nature, 1992; 355: 827-829
[23] Fletcher J I, Smith R O, Donoghue S I, Nilges M, Connor M et al. Nat. Strut. Biol, 1997, 4: 559-566
[24] Little M J, Wilson H, Zappia C, Cestle Set al. FEBS Lett, 1998 Nov; 439(3): 246-452
[25] Szeto T H, Wang X H, Smith R, Connor M, Christie M. J, NicholsonG M, King G F. Toxin, 2000 Mar; 38(3): 429-442
[26] Haginwara K, Sakai T, Miwa K et al. FEBS Letts, 1990: 11: 2181
[27] Diniz C R, Cordeiro M N, Janor L R et al. FEBS Letts, 1990; 263: 251
[28] Reis H J, Prado M A et al. Biochem J, 1999; 343 pt 2: 413-418
[29] Santos R G, Diniz C R, Cordeiro M N, De Lima M E. Braz J Med Biol Res 1999 Dec; 32(12): 1565-9 '
[30] Cordeiro M N, Diniz C R, Cordeiro M N, De Lima M E. graz J Med Biol Res 1999 Dec; 32(12): 1565-1569
[31] Troncone J R, Lebrun I, Magnoli F, Yamane T. Neurochem Res, 1995 Jul; 20(7): 879-883
[32] Lampe R A, Defeo P A et al. Mol Pharmacol 1993; 44: 451-460
[33] Swartz K J&Mackion R. Neuro, 1995; 15: 941-949
[34] Li-Smerin Y&Swartz K J. Proc Natl Acad Sci USA, 1998; 95: 8585-8589
[35] Newcomb R, SzokeB. etal. Biochemistry, 1998; 37: 15353-15362
[36] Skinner W S, Dennis P A, JorgePLietal. Toxicon, 1992; 30(9): 1043
[37] 梁宋平，覃于滨，张东裔等．动物学研究，1993；14(1)：60
[38] Liang S P, Zhang D Y, PanX et al. Toxicon, 1993; 31: 969
[39] ZhangD Y, Liang S P J Protein Cchem, 1993; 12(6): 733
[40] Qu Y X, Liang S P, Liu X, Zhang R and Gu X. J Pretein
Chem, 1997; 16: 565-574
[41] Zhou P A, Xie X J, Li M et al. Toxicon, 1997; 35: 39-45
[42] 梁宋平，夏赞贤，谢锦云．中国科学(辑)，1997：27(5)，391—397
[43] 徐科，施玉梁，吉永华，王文萍．生理通讯(增刊号)，1986：第一页
[44] 徐科，吉永华，屈贤铭．动物学报，1989：35(3)
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[46] Sanguinetti M C, Jonhson JH et al. Mol Pharmacol, 1997; 52: 491-498
[47] MarvinL, De E, Cosette P et al. Eur J Biochem, 1999 Oct; 265(2): 572-579
[48] Desai A., Miller M J., Gomez H F. Etal; J Toxicol Clin Toxicol, 1999, 37(4): 447-456
[49] Desai A, LandfordHA, WARRAN J S, Infelammation, 2000 Feb; 24(1): 1-9
[50] Ficher FG and Bohn H. Justus Liebigs Annalender Vhemmie, 1957; 603: 232-250
[51] Grishin E V, VolkovaTM et al. Bio-org Khim, 1986; 12: 1121-1124
[52] Bateman A et al. Brain Res, 1985; 339: 237-244
[53] Budd T et al. Brain Res, 1988; 448: 30-39
[54] Kerry C J, Ramsey R I, Sansom M S P, Usherwood P N R. Brain Res, 1988; 459: 312-327
[55] Ashe J H, Cox C L, Adams M E Brain Res, 1989; 234-241
[56] Donevan S D, Rogawski M A. Neuroscience, 1996; 70: 361-375
[57] Piestly T, WoodruffGN, Kemp J A. Br J Pharmacol, 1989; 97: 1315-1323
[58] Herliteze S, Raditsch M et al. Neuron, 1993; 10: 1131-1140
[59] Reynold I J. Br J Pharmacol, 1991; 103: 1131-1140
[60] Williams K. J Pharmacol Exp Ther, 1993; 266: 231-236
[61] Raditsch M, Geyer M et al. Eur J Biochem, 1996; 240: 3: 37-43
[62] Sutten K G, Dolphin A C, Scott R H. Mol Neuropharmacol, 1993; 3: 37-43
[63] Scott, R, H, Satton, K G, Dolphin A C, Academic Press, 1994; 205-222
[64] Llina R R, Sugimori M, Hillman D E and Cherksey, B. Trends Neurosci, 1992; 15: 351-355
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[68] Reis H J, Prodo M A, Kalapothadis E, Cordeiro M N, Qinic CRm, Qe Marco LA, Gomez M V Romane Silva M A. Biochem J 1999 Oct. 15; 343Pt 2: 413-418
[69] Toxin 30, 303-322, 1992
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[71] Masayuki Matsushita, Takanori Kanemura, Susml. Hatakey ama, Hiroshi Irie, Takashi Toki, and Masaaki Miyashita. Tetrahedr on Letters Vol 36. No. 29. PP. 5231-5234, 1995
[72] Roder H. Scott, Kathy G, Sutton and Annette C, Qolphin. Trends in NewroSciences. April, 1993
[2] 陈远聪，生命的化学，1991；11(3)：1-2
[3] 王贤纯，博士毕业论文，1998
[4] Eugene. Grinshin, Eur. J. Biochem, 1999; 264: 276-280
[5] 梁宋平，潘欣。生物化学与生物物理进展，1994；21(5)：390—395
[6] Longenecker H E Tr, Hurlbut W P, Mauro A et al. Nature, 1970; 225: 701
[7] Frontail N, Ceccarelli B, Gorio A et al. J Cell Biol, 1976: 68: 462
[8] Hurlbut. W P., Iezzi. N, Fesce. R. &Ceccarelli. B, J. Physiol, 1990; 425: 501-526
[9] Petrenko A G, Kovalenko V A, Shamotienko Z N et al. EMBO Journal, 1990; 9: 2023
[10] Kiyatkin N I, Dulubova I E, Cheknovskaya I A et al. FEBS Letts, 1990; 270: 127
[11] Lurnev, A. V., Oemin. V. V. etal, Bioong. Khim, 1991; 171021-1026
[12] Erishin E V. Toxicon, 1998 Nov; 36(11): 1693-701
[13] Michaely. p. &Bennett. VTrends, Cell Biol, 1992; 2: 127-129
[14] Danilevich V N, Lukianov S A, Grishin E V. Bioorg Khim. 1999 Jul; 25(7): 537-547
[15] Sheumack D D, Classens R, Whiteleg N M et al. FEBS Letts, 1985; 181: 154
[16] Brown M R, Sheumack D D, Tyler M I et al. Biochem J, 1988; 250: 401
[17] Nicholson G M, Little M J, Tyler M, Narahashi T. Toxicon, 1996 Nov-Dec; 34(11-12): 1443-1453
[18] Skinner W S, Adams M E, Quistad et al. J, Biol Chem, 1989; 264: 2150
[19] Adams M E, Bindokas V P, Hasewage L&Veneva V J. J Biol Chem, 1990; 265: 861-867
[20] Ertel E A, Waren V A, Adams M E, Griffin P R etal. Bioc
hemistry, 1994; 33: 5098-5208
[21] Mintz I M, Venema V J, Adams M E&Bean B P. Proc. Natl. A cad. Sci. USA, 1991; 88: 6628-6631
[22] Mintz I M Venema V J, Swiderek K M et al. Nature, 1992; 355: 827-829
[23] Fletcher J I, Smith R O, Donoghue S I, Nilges M, Connor M et al. Nat. Strut. Biol, 1997, 4: 559-566
[24] Little M J, Wilson H, Zappia C, Cestle Set al. FEBS Lett, 1998 Nov; 439(3): 246-452
[25] Szeto T H, Wang X H, Smith R, Connor M, Christie M. J, NicholsonG M, King G F. Toxin, 2000 Mar; 38(3): 429-442
[26] Haginwara K, Sakai T, Miwa K et al. FEBS Letts, 1990: 11: 2181
[27] Diniz C R, Cordeiro M N, Janor L R et al. FEBS Letts, 1990; 263: 251
[28] Reis H J, Prado M A et al. Biochem J, 1999; 343 pt 2: 413-418
[29] Santos R G, Diniz C R, Cordeiro M N, De Lima M E. Braz J Med Biol Res 1999 Dec; 32(12): 1565-9 '
[30] Cordeiro M N, Diniz C R, Cordeiro M N, De Lima M E. graz J Med Biol Res 1999 Dec; 32(12): 1565-1569
[31] Troncone J R, Lebrun I, Magnoli F, Yamane T. Neurochem Res, 1995 Jul; 20(7): 879-883
[32] Lampe R A, Defeo P A et al. Mol Pharmacol 1993; 44: 451-460
[33] Swartz K J&Mackion R. Neuro, 1995; 15: 941-949
[34] Li-Smerin Y&Swartz K J. Proc Natl Acad Sci USA, 1998; 95: 8585-8589
[35] Newcomb R, SzokeB. etal. Biochemistry, 1998; 37: 15353-15362
[36] Skinner W S, Dennis P A, JorgePLietal. Toxicon, 1992; 30(9): 1043
[37] 梁宋平，覃于滨，张东裔等．动物学研究，1993；14(1)：60
[38] Liang S P, Zhang D Y, PanX et al. Toxicon, 1993; 31: 969
[39] ZhangD Y, Liang S P J Protein Cchem, 1993; 12(6): 733
[40] Qu Y X, Liang S P, Liu X, Zhang R and Gu X. J Pretein
Chem, 1997; 16: 565-574
[41] Zhou P A, Xie X J, Li M et al. Toxicon, 1997; 35: 39-45
[42] 梁宋平，夏赞贤，谢锦云．中国科学(辑)，1997：27(5)，391—397
[43] 徐科，施玉梁，吉永华，王文萍．生理通讯(增刊号)，1986：第一页
[44] 徐科，吉永华，屈贤铭．动物学报，1989：35(3)
[45] 赵维勤，吉永华，徐科等．动物学研究，1991：12(1)
[46] Sanguinetti M C, Jonhson JH et al. Mol Pharmacol, 1997; 52: 491-498
[47] MarvinL, De E, Cosette P et al. Eur J Biochem, 1999 Oct; 265(2): 572-579
[48] Desai A., Miller M J., Gomez H F. Etal; J Toxicol Clin Toxicol, 1999, 37(4): 447-456
[49] Desai A, LandfordHA, WARRAN J S, Infelammation, 2000 Feb; 24(1): 1-9
[50] Ficher FG and Bohn H. Justus Liebigs Annalender Vhemmie, 1957; 603: 232-250
[51] Grishin E V, VolkovaTM et al. Bio-org Khim, 1986; 12: 1121-1124
[52] Bateman A et al. Brain Res, 1985; 339: 237-244
[53] Budd T et al. Brain Res, 1988; 448: 30-39
[54] Kerry C J, Ramsey R I, Sansom M S P, Usherwood P N R. Brain Res, 1988; 459: 312-327
[55] Ashe J H, Cox C L, Adams M E Brain Res, 1989; 234-241
[56] Donevan S D, Rogawski M A. Neuroscience, 1996; 70: 361-375
[57] Piestly T, WoodruffGN, Kemp J A. Br J Pharmacol, 1989; 97: 1315-1323
[58] Herliteze S, Raditsch M et al. Neuron, 1993; 10: 1131-1140
[59] Reynold I J. Br J Pharmacol, 1991; 103: 1131-1140
[60] Williams K. J Pharmacol Exp Ther, 1993; 266: 231-236
[61] Raditsch M, Geyer M et al. Eur J Biochem, 1996; 240: 3: 37-43
[62] Sutten K G, Dolphin A C, Scott R H. Mol Neuropharmacol, 1993; 3: 37-43
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