东亚马氏钳蝎中新型活性多肽的纯化，基因克隆与功能鉴定
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摘要
在漫长的3.5亿年的生存选择中，蝎子逐渐进化出了形形色色，功能各异的神经活性多肽。这些多肽是它们进攻与防御的主要武器，大多作用于各类Na~+和K~+通道。K~+通道是生物体中最基础的功能蛋白之一，在诸多的生理过程中起着至关重要的作用。目前，蝎活性多肽已经成为研究K~+通道的药理，生理，结构和功能特性的最有力的工具。然而K~+通道成员复杂，含有80多种亚型，其中许多亚型的性质几乎是一无所知。为了得到更多研究K~+通道的工具，本文致力于从东亚马氏钳蝎中的毒腺中纯化和鉴定新型K~+通道的阻断剂。
通过综合运用各类色谱技术，本文共纯化和测序了5个新型K~+通道的阻断剂：BmBKTx1，BmTx3，BmSKTx1，BmSKTx2和BmK38。同时BmSKTx1，BmSKTx2和BmK38的cDNA基因以及BmSKTx1的Genomic基因也被分别克隆。功能鉴定的结果表明BmBKTx1是一个钙激活型钾离子通道(K_(Ca))的阻断剂，能同时作用于高电导型(BK)和低电导型(SK)的K_(Ca)通道。在种属的选择性上，BmBKTx1只作用于昆虫的BK通道。BmTx3是一个双功能多肽，分子上具有两个分离的功能面，分别对应于HERG通道和A型钾电流的阻断活性。BmSKTx1和BmSKTx2是专一的SK通道阻断剂。BmK38的生理功能还未有明确的阐述。从一级结构上看，它属于K~+通道阻断剂这一大类。从高级结构上看，它有一个明显突出于分子表面的β转角，暗示了其功能的特异性。
中文摘要
本文同时从东亚马氏钳蝎中纯化到了一个Chlorotoxin的类似物
BmCITx。Chiorotoxin是一个特异性抑制神经胶质瘤的药物，正处于班I期
临床试验。因此BmCITxl的纯化也提供了一个潜在的抗肿瘤药物前体。
除了各种实验方法外，本文还运用流行的进化痕迹分析法综合分析了各
类K+通道阻断剂在进化上的保守性和趋异性，预测了作用于刀石尺G通道的
Y--KTx的功能面，并进一步详细讨论了孔区阻断与塔楼区阻断这两种阻断方
式的区别。
关键词:蝎活性多肤，钾离子通道，基因克隆，电生理，进化痕迹分析
As one of the most fundamental proteins in organisms, K+ channels play key roles in many crucial physiological processes. So far, more than 80 K+ channels have been identified but many of them are poorly characterized. Scorpions have survived more than 350 million years with no detectable changes in their anatomy due to their efficient, dynamic machinery production of bioactive peptides mainly blocking Na+ channels and K+ channels. Scorpion peptides have been proven to be powerful tools for testing the pharmacological, physiological, structural and functional characteristics of K+ channels. To get more tools for K+ channel studies, it is important to keep searching for novel bioactive peptides from scorpion venom.In the present study, five novel K+ channel blockers, namely BmBKTxl, BmTx3, BmSKTxl, BmSKTx2 and BmK38, have been purified from the crude venom of Scorpion Buthus martensi Karsh (BmK). Based on their determined protein sequences, gene special primers were designed and the cDNA genes of BmSKTxl, BmSKTx2 and BmK38 were cloned by Rapid Amplification of cDNA Ends (RACE). Also, the genomic gene of BmSKTxl was cloned by PCR. After screened on various channels, BmBKTxl was turned out to be a selective blocker for Ca2+-activated K+ channels, with the activity both on BK-type (big conductance) and SK-type (small conductance) KCa channels. Interestingly, BmBKTxl tends to have the insect specificity in respect of the BK channel blocking. BmTx3 is a peptide with two different functions: HERG channel and A-
type K+ current blocking activities. There are two separate functional faces locating on the N-terminal region and the C-terminal region of the BmTx3 molecule, respectively corresponding to its two different K+ currents blocking abilities. BmSKTxl and BmSKTx2 are selective blockers for SK channels. Although the function of BmK38 has not been clearly demonstrated, it has the same cysteine scaffold shared by other K+ channel blockers, indicating that it should target K+ channels also. However, there is a unique turn sticking out from the whole molecule surface of BmK38, implying that this toxin might have a unique function.A chlorotoxin analogue, designated BmClTx, was also purified from scorpion BmK. Chlorotoxin is a selective drug to treat gliomas and has recently won the FDA approval for use in a phase I/II clinical trial. Given the high sequence homology between BmClTxl and Chlorotoxin, BmClTx also holds the potential to be developed to an anti-cancer drug.Besides various experimental methods, a computational method, Evolutionary trace (ET) analysis, was performed to study the evolutionary convergence and divergence among different scorpion K+ channels blockers. The functional face of y-KTx active on HERG channel was predicted by ET analysis and the differences between two block modes - turret block and pore block- were fully discussed in this study.
通过综合运用各类色谱技术，本文共纯化和测序了5个新型K~+通道的阻断剂：BmBKTx1，BmTx3，BmSKTx1，BmSKTx2和BmK38。同时BmSKTx1，BmSKTx2和BmK38的cDNA基因以及BmSKTx1的Genomic基因也被分别克隆。功能鉴定的结果表明BmBKTx1是一个钙激活型钾离子通道(K_(Ca))的阻断剂，能同时作用于高电导型(BK)和低电导型(SK)的K_(Ca)通道。在种属的选择性上，BmBKTx1只作用于昆虫的BK通道。BmTx3是一个双功能多肽，分子上具有两个分离的功能面，分别对应于HERG通道和A型钾电流的阻断活性。BmSKTx1和BmSKTx2是专一的SK通道阻断剂。BmK38的生理功能还未有明确的阐述。从一级结构上看，它属于K~+通道阻断剂这一大类。从高级结构上看，它有一个明显突出于分子表面的β转角，暗示了其功能的特异性。
中文摘要
本文同时从东亚马氏钳蝎中纯化到了一个Chlorotoxin的类似物
BmCITx。Chiorotoxin是一个特异性抑制神经胶质瘤的药物，正处于班I期
临床试验。因此BmCITxl的纯化也提供了一个潜在的抗肿瘤药物前体。
除了各种实验方法外，本文还运用流行的进化痕迹分析法综合分析了各
类K+通道阻断剂在进化上的保守性和趋异性，预测了作用于刀石尺G通道的
Y--KTx的功能面，并进一步详细讨论了孔区阻断与塔楼区阻断这两种阻断方
式的区别。
关键词:蝎活性多肤，钾离子通道，基因克隆，电生理，进化痕迹分析
As one of the most fundamental proteins in organisms, K+ channels play key roles in many crucial physiological processes. So far, more than 80 K+ channels have been identified but many of them are poorly characterized. Scorpions have survived more than 350 million years with no detectable changes in their anatomy due to their efficient, dynamic machinery production of bioactive peptides mainly blocking Na+ channels and K+ channels. Scorpion peptides have been proven to be powerful tools for testing the pharmacological, physiological, structural and functional characteristics of K+ channels. To get more tools for K+ channel studies, it is important to keep searching for novel bioactive peptides from scorpion venom.In the present study, five novel K+ channel blockers, namely BmBKTxl, BmTx3, BmSKTxl, BmSKTx2 and BmK38, have been purified from the crude venom of Scorpion Buthus martensi Karsh (BmK). Based on their determined protein sequences, gene special primers were designed and the cDNA genes of BmSKTxl, BmSKTx2 and BmK38 were cloned by Rapid Amplification of cDNA Ends (RACE). Also, the genomic gene of BmSKTxl was cloned by PCR. After screened on various channels, BmBKTxl was turned out to be a selective blocker for Ca2+-activated K+ channels, with the activity both on BK-type (big conductance) and SK-type (small conductance) KCa channels. Interestingly, BmBKTxl tends to have the insect specificity in respect of the BK channel blocking. BmTx3 is a peptide with two different functions: HERG channel and A-
type K+ current blocking activities. There are two separate functional faces locating on the N-terminal region and the C-terminal region of the BmTx3 molecule, respectively corresponding to its two different K+ currents blocking abilities. BmSKTxl and BmSKTx2 are selective blockers for SK channels. Although the function of BmK38 has not been clearly demonstrated, it has the same cysteine scaffold shared by other K+ channel blockers, indicating that it should target K+ channels also. However, there is a unique turn sticking out from the whole molecule surface of BmK38, implying that this toxin might have a unique function.A chlorotoxin analogue, designated BmClTx, was also purified from scorpion BmK. Chlorotoxin is a selective drug to treat gliomas and has recently won the FDA approval for use in a phase I/II clinical trial. Given the high sequence homology between BmClTxl and Chlorotoxin, BmClTx also holds the potential to be developed to an anti-cancer drug.Besides various experimental methods, a computational method, Evolutionary trace (ET) analysis, was performed to study the evolutionary convergence and divergence among different scorpion K+ channels blockers. The functional face of y-KTx active on HERG channel was predicted by ET analysis and the differences between two block modes - turret block and pore block- were fully discussed in this study.
引文
1 Rodriguez de la Vega, R. C, Merino, E., Becerril, B., and Possani, L. D. (2003) Novel interactions between K+ channels and scorpion toxins. Trends Pharmacol. Sci. 24, 222-7
2 Wickenden, A. (2002) K(+) channels as therapeutic drug targets. Pharmacol. Ther. 94, 157-82
3 Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69-77
4 Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T., and MacKinnon, R. (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423, 33-41
5 Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies. Trends Pharmacol. Sci. 20, 444-7
6 M'Barek, S., Lopez-Gonzalez, I., Andreotti, N., di Luccio, E., Visan, V., Grissmer, S., Judge, S., El Ayeb, M., Darbon, H., Rochat, H., Sampieri, F., Beraud, E., Fajloun, Z., De Waard, M., and Sabatier, J. M. (2003) A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis, pharmacology, and docking on K+ channels. J. Biol. Chem. 278, 31095-104
7 Corona, M., Gurrola, G. B., Merino, E., Cassulini, R. R., Valdez-Cruz, N. A., Garcia, B., Ramirez-Dominguez, M. E., Coronas, F. I., Zamudio, F. Z., Wanke, E., and Possani, L. D. (2002) A large number of novel Ergtoxin-like genes and ERG K+-channels blocking peptides from scorpions of the genus Centruroides. FEBS Lett. 532, 121-6
8 Curran, M. E., Splawski, L, Timothy, K. W., Vincent, G. M., Green, E. D., and Keating, M. T. (1995) A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80, 795-803
9 Korolkova, Y. V., Kozlov, S. A., Lipkin, A. V., Pluzhnikov, K. A., Hadley, J. K., Filippov, A. K., Brown, D. A., Angelo, K., Strobaek, D., Jespersen, T., Olesen, S. P., Jensen, B. S., and Grishin, E. V. (2001) An ERG channel inhibitor from the scorpion Buthus eupeus. J. Biol. Chem. 276, 9868-76
10 Legros, C, Ceard, B., Bougis, P. E., and Martin-Eauclaire, M. F. (1998) Evidence for a new class of scorpion toxins active against K+ channels. FEBS Lett. 431, 375-80
11 Froy, O., and Gurevitz, M. (2003) New insight on scorpion divergence inferred from comparative analysis of toxin structure, pharmacology and distribution. Toxicon 42, 549-55
12 Srinivasan K.N., Sivaraja V., Huys I., Sasaki T., Cheng B., Kumar T.K., Sato K., Tytgat J., Yu C, San B.C., Ranganathan S., Bowie H.J., Kini R.M., and Gopalakrishnakone P. (2002) kappa-Hefutoxinl, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity. J. Biol. Chem. 277, 30040-7
13 Krezel, A. M., Kasibhatla, C, Hidalgo, P., MacKinnon, R., and Wagner, G. (1995) Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry. Protein Sci. 4, 1478-89
14 Torres, A. M., Bansal, P., Alewood, P. F., Bursill, J. A., Kuchel, P. W., and Vandenberg, J. I. (2003) Solution structure of CnErgl (Ergtoxin), a HERG specific scorpion toxin. FEBS Lett. 539, 138-42
15 Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, B., Roumestand, C, Vita, C, de Medeiros, C. L., Rowan, E. G., Harvey, A. L., and Menez, A. (1997) On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J. Biol. Chem. 272, 4302-9
16 Huys, I., Olamendi-Portugal, T., Garcia-Gomez, B. L, Vandenberghe, L, Van Beeumen, J., Dyason, K., Clynen, E., Zhu, S., Van Der Walt, J., Possani, L., and Tytgat, J. (2004) A subfamily of acidic alpha -K+ toxins. J. Biol. Chem. 279, 2781-9
17 Mouhat, S., Mosbah, A., Visan, V., Wulff, H., Delepierre, M., Darbon, H., Grissmer, S., De Waard, M., and Sabatier, J. M. (2003) The "functional" dyad of scorpion toxin Pil is not per se a prerequisite for toxin binding to the voltage-gated Kvl.2 potassium channels. Biochem J. Ptl, 25-36
18 Wang, C. G., Grilles, N., Hamon, A., Le Gall, F., Stankiewicz, M., Pelhate, M., Xiong, Y. M., Wang, D. C, and Chi, C. W. (2003) Exploration of the functional site of a scorpion alpha-like toxin by site-directed mutagenesis. Biochemistry 42, 4699-708
19 Gurevitz, M., Gordon, D., Ben-Natan, S., Turkov, M., and Froy, O. (2001) Diversification of neurotoxins by C-tail 'wiggling': a scorpion recipe for survival. FASEB J. 15, 1201-5.
20 DeBin, J. A., Maggio, J. E., and Strichartz, G. R. (1993) Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am. J. Physiol. 264, C361-9
21 Soroceanu, L., Gillespie, Y., Khazaeli, M. B., and Sontheimer, H. (1998) Use of chlorotoxin for targeting of primary brain tumors. Cancer Res. 58, 4871-9
22 Maertens, C, Wei, L., Tytgat, J., Droogmans, G., and Nilius, B. (2000) Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels. Br. J. Pharmacol. 129, 791-801
23 Zamudio, F. Z., Gurrola, G. B., Arevalo, C, Sreekumar, R., Walker, J. W., Valdivia, H. H., and Possani, L. D. (1997) Primary structure and synthesis of Imperatoxin A (IpTx(a)), a peptide activator of Ca2+ release channels/ryanodine receptors. FEBS Lett. 405, 385-9
24 Fajloun, Z., Kharrat, R., Chen, L., Lecomte, C, Di Luccio, E., Bichet, D., El Ayeb, M., Rochat, H., Allen, P. D., Pessah, I. N., De Waard, M., and Sabatier, J. M. (2000) Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca(2+) release channel/ryanodine receptors. FEBS Lett. 469, 179-85
25 Garcia, M. L., Gao, Y., McManus, O. B., and Kaczorowski, G. J. (2001) Potassium channels: from scorpion venoms to high-resolution structure. Toxicon 39, 739-48
26 Vita, C, Drakopoulou, E., Vizzavona, J., Rochette, S., Martin, L., Menez, A., Roumestand, C, Yang, Y. S., Ylisastigui, L., Benjouad, A., and Gluckman, J. C. (1999) Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein. Proc. Natl. Acad. Sci. USA 96, 13091-6
2 Wickenden, A. (2002) K(+) channels as therapeutic drug targets. Pharmacol. Ther. 94, 157-82
3 Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69-77
4 Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T., and MacKinnon, R. (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423, 33-41
5 Tytgat, J., Chandy, K. G., Garcia, M. L., Gutman, G. A., Martin-Eauclaire, M. F., van der Walt, J. J., and Possani, L. D. (1999) A unified nomenclature for short-chain peptides isolated from scorpion venoms: alpha-KTx molecular subfamilies. Trends Pharmacol. Sci. 20, 444-7
6 M'Barek, S., Lopez-Gonzalez, I., Andreotti, N., di Luccio, E., Visan, V., Grissmer, S., Judge, S., El Ayeb, M., Darbon, H., Rochat, H., Sampieri, F., Beraud, E., Fajloun, Z., De Waard, M., and Sabatier, J. M. (2003) A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis, pharmacology, and docking on K+ channels. J. Biol. Chem. 278, 31095-104
7 Corona, M., Gurrola, G. B., Merino, E., Cassulini, R. R., Valdez-Cruz, N. A., Garcia, B., Ramirez-Dominguez, M. E., Coronas, F. I., Zamudio, F. Z., Wanke, E., and Possani, L. D. (2002) A large number of novel Ergtoxin-like genes and ERG K+-channels blocking peptides from scorpions of the genus Centruroides. FEBS Lett. 532, 121-6
8 Curran, M. E., Splawski, L, Timothy, K. W., Vincent, G. M., Green, E. D., and Keating, M. T. (1995) A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80, 795-803
9 Korolkova, Y. V., Kozlov, S. A., Lipkin, A. V., Pluzhnikov, K. A., Hadley, J. K., Filippov, A. K., Brown, D. A., Angelo, K., Strobaek, D., Jespersen, T., Olesen, S. P., Jensen, B. S., and Grishin, E. V. (2001) An ERG channel inhibitor from the scorpion Buthus eupeus. J. Biol. Chem. 276, 9868-76
10 Legros, C, Ceard, B., Bougis, P. E., and Martin-Eauclaire, M. F. (1998) Evidence for a new class of scorpion toxins active against K+ channels. FEBS Lett. 431, 375-80
11 Froy, O., and Gurevitz, M. (2003) New insight on scorpion divergence inferred from comparative analysis of toxin structure, pharmacology and distribution. Toxicon 42, 549-55
12 Srinivasan K.N., Sivaraja V., Huys I., Sasaki T., Cheng B., Kumar T.K., Sato K., Tytgat J., Yu C, San B.C., Ranganathan S., Bowie H.J., Kini R.M., and Gopalakrishnakone P. (2002) kappa-Hefutoxinl, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity. J. Biol. Chem. 277, 30040-7
13 Krezel, A. M., Kasibhatla, C, Hidalgo, P., MacKinnon, R., and Wagner, G. (1995) Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry. Protein Sci. 4, 1478-89
14 Torres, A. M., Bansal, P., Alewood, P. F., Bursill, J. A., Kuchel, P. W., and Vandenberg, J. I. (2003) Solution structure of CnErgl (Ergtoxin), a HERG specific scorpion toxin. FEBS Lett. 539, 138-42
15 Dauplais, M., Lecoq, A., Song, J., Cotton, J., Jamin, N., Gilquin, B., Roumestand, C, Vita, C, de Medeiros, C. L., Rowan, E. G., Harvey, A. L., and Menez, A. (1997) On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J. Biol. Chem. 272, 4302-9
16 Huys, I., Olamendi-Portugal, T., Garcia-Gomez, B. L, Vandenberghe, L, Van Beeumen, J., Dyason, K., Clynen, E., Zhu, S., Van Der Walt, J., Possani, L., and Tytgat, J. (2004) A subfamily of acidic alpha -K+ toxins. J. Biol. Chem. 279, 2781-9
17 Mouhat, S., Mosbah, A., Visan, V., Wulff, H., Delepierre, M., Darbon, H., Grissmer, S., De Waard, M., and Sabatier, J. M. (2003) The "functional" dyad of scorpion toxin Pil is not per se a prerequisite for toxin binding to the voltage-gated Kvl.2 potassium channels. Biochem J. Ptl, 25-36
18 Wang, C. G., Grilles, N., Hamon, A., Le Gall, F., Stankiewicz, M., Pelhate, M., Xiong, Y. M., Wang, D. C, and Chi, C. W. (2003) Exploration of the functional site of a scorpion alpha-like toxin by site-directed mutagenesis. Biochemistry 42, 4699-708
19 Gurevitz, M., Gordon, D., Ben-Natan, S., Turkov, M., and Froy, O. (2001) Diversification of neurotoxins by C-tail 'wiggling': a scorpion recipe for survival. FASEB J. 15, 1201-5.
20 DeBin, J. A., Maggio, J. E., and Strichartz, G. R. (1993) Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am. J. Physiol. 264, C361-9
21 Soroceanu, L., Gillespie, Y., Khazaeli, M. B., and Sontheimer, H. (1998) Use of chlorotoxin for targeting of primary brain tumors. Cancer Res. 58, 4871-9
22 Maertens, C, Wei, L., Tytgat, J., Droogmans, G., and Nilius, B. (2000) Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels. Br. J. Pharmacol. 129, 791-801
23 Zamudio, F. Z., Gurrola, G. B., Arevalo, C, Sreekumar, R., Walker, J. W., Valdivia, H. H., and Possani, L. D. (1997) Primary structure and synthesis of Imperatoxin A (IpTx(a)), a peptide activator of Ca2+ release channels/ryanodine receptors. FEBS Lett. 405, 385-9
24 Fajloun, Z., Kharrat, R., Chen, L., Lecomte, C, Di Luccio, E., Bichet, D., El Ayeb, M., Rochat, H., Allen, P. D., Pessah, I. N., De Waard, M., and Sabatier, J. M. (2000) Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca(2+) release channel/ryanodine receptors. FEBS Lett. 469, 179-85
25 Garcia, M. L., Gao, Y., McManus, O. B., and Kaczorowski, G. J. (2001) Potassium channels: from scorpion venoms to high-resolution structure. Toxicon 39, 739-48
26 Vita, C, Drakopoulou, E., Vizzavona, J., Rochette, S., Martin, L., Menez, A., Roumestand, C, Yang, Y. S., Ylisastigui, L., Benjouad, A., and Gluckman, J. C. (1999) Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein. Proc. Natl. Acad. Sci. USA 96, 13091-6