摘要
1：虎纹捕鸟蛛(Ornithochoctonus huwena)是分布在我国西南部山区的一种蜘蛛新种。它的粗毒中含有多种活性成分，其中研究的比较深入的多为神经毒素，如HWTX-Ⅰ，这是一种突触前膜N型钙离子通道阻断剂；毒素HWTX-Ⅳ，它是一种TTX敏感型钠离子通道阻断剂；另外，还从虎纹粗毒中纯化出一种具有凝集活性毒素成分SHL-Ⅰ。
本文报道从虎纹捕鸟蛛(Ornithochoctonus huwena)粗毒中，应用阳离子交换和反相高效液相色谱的方法分离到一种胰蛋白酶抑制剂，命名为Huwentoxin-Ⅺ(HWTX-Ⅺ)。经MALDI-TOF质谱技术鉴定，HWTX-Ⅺ的相对分子质量为6166.2。经DTT还原，进一步用碘乙酰胺修饰其相对分子质量增加到6514.2，根据一个半胱氨酸修饰相对分子质量增加58，我们可以推断HWTX-Ⅺ有三对二硫键。利用Edman降解气相蛋白质测序仪测得HWTX-Ⅺ的一级结构NH2-IDTCRLPSDRGRCKASFERWYFNGRTCAKFIYGGCGGNGNKFPTQEACMKRCAKA-COOH，理论相对分子质量比质谱测得的值多6，由此也可以确定六个半胱氨酸形成了三对二硫键。利用传统的酶促动力学研究方法通过分光光度计测定HWTX-Ⅺ对胰蛋白酶的抑制活性发现HWTX-Ⅺ对胰蛋白酶有很强的抑制作用，完全抑制的摩尔抑制比为1：1。用Lineveaer-Burk作图法得知，该抑制剂属竞争性抑制类型，具ki值为2.938×10~(-8)mol/L(以BAPNA为底物)。用BIAcore X测定该毒素和胰蛋白酶及胰凝乳蛋白酶的生物大分子相互作用，同样发现HWTX-Ⅺ对胰蛋白酶有很强的抑制作用，对胰凝乳蛋白酶也有中等程度的抑制作用，解离常数Kd分别为1.23×10~(-10)mol／L和1.14×10~(-7)mol/L。HWTX-Ⅺ对昆明种小白鼠第四脑室注射表现出较强的活性，其半致死量LD_(50)为256ug／kg。由此可见，HWTX-Ⅺ也是一种哺乳动物中枢神经毒素。
Abstract l:The Chinese bird spider, Ornithochoctonus huwena, distributed in the hilly areas of Yunnan and Guangxi in the south of China, was recently identified as a new species and is one of the most venomous spiders in China. In our previous work, we have demonstrated that 0. huwena venom contains a mixture of compounds with different types of biological activities. Most of them are neurotoxins, such as HWTX-1, -II ,-III, IV.
Huwentoxin XI (HWTX~XI),a serine protease! inhibitor, consists of 55 amino acid residues with three disulfide bridges. The toxin was isolated from the venom of the Chinese spider Ornithochoctonus huwena by ion-exchange chromatogram -phy and reverse phase high performance liquid chromatography.
The jmolecular weight of HWTX-XI is 6166. 2, determined by MALDI-TOF mass spectrometry ,which is identical with the
calculated mass based on the complete amino acid sequence of NH2-IDTCRLPSDRGRCKASFERWYFNGRTCAKFIYGGCGGNGNKFPTQEACMCAKA - COOH, determined by automatic Edman degradeation .The inhibition of trypsin by HWTX-XI was competetive ,with a ki value of 2. 938 X 108mol/L (BAPNA as substrate), which is determined by using a U-2000 spectrophotometer instrument (HITACHI , Japan) .The binding properties of HWTX-XI on trypsin and a -chymotrypsin were measured by a BIAcore binding assay system. Results showed that HWTX-XI could bind to trypsin with a dissociation constant of 1. 23 X 10-10mol/L, and to a -chymotrypsin with a dissociation constant of 1. 14 X 10-7mol/L . Inhibition profiles of trypsin by HWTX-XI revealed a 1:1 binding stoichiometry between this inhibitor and trypsin.
Huwentoxin XI also have a strong biological activity with a LD5n=256 u g/kg when injected into the fourth ventricle of the adult mouse brain.
引文
[1] Scott, R. H., Sutton, K. G., Annette, C. D. Trends in Neurosciencecs. 1993; 16(4): 153-159.
[2] Hulbut, W. P., Lezzi, N., Fesce, R., Ceccarelli, B. J. Physiol. 1990; 425: 501-526.
[3] CZAPINSKA, H. OTLEWSKI, J. KRZYWDA, S. High resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence. J. MOL. Biol. 2000, 1237-1249
[4] Petrenko, A. G., Kovalenko, V. A. Shamotienko, Z. N., et al. EMBO Joural. 1990; 9: 2023.
[5] Lurnev, A. V., Qemin, V. V., et al. Bioong Khim. 1991; 17: 1021-26.
[6] nanilevich, V. N., Luk' ianov, S. A., Grishin, E. Y. Bioorg Khim. 1999; 25(7): 537-547.
[7] Sheumack, D. D., Classens, R. Whiteleg, N. M., et al. FEBS Letters. 1985; 181: 154.
[8] Brown, M. R., Sheumack, D. D., Tyler, M. I., et al. Biochem. J. 1988; 250: 401.
[9] Nicholson, G. M., Little, M. J., Tyler, M., Narahashi, T. Toxicon. 1996; 34(11-12): 1443-53.
[10] SCHEIDIG AJ, HYNES TR, PELLETIER LA, WELLS JA, KOSSIAKOFF AA. Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered. Protein Sci 1997 (9): 1806-24
[11] Admas, M. E., Binokas, V. P., Hasewage, L., Veneva, V. J. J. Biol. Chem. 1990; 265: 861-867.
[12] Ertel, E. A., Waren, V. J., Adams, M. E., et al., Biochemstry. 1994; 33: 5098-5108.
[13] Fletcher, J. I., Smith, R. O., Donoghue, S. I., Nilges, M., Connor, M., et al., Nat. Struct. Bio. 1997; 4: 559-566.
[14] Little, M. J., Wilson, H., Zappia, C., Cestle, S., et al., FEBS
Letter. 1998; 439(3): 246-52.
[15] Rash, L. D., Hodgson, W. C., Toxicon. 2002; 40: 225-254.
[16] Escoubas, P., Diochot, S., Gerardo, C., Biochimie. 2000; 82: 893-907.
[17] Young, A. R., Pincus, S. J., Toxicon. 2001; 39: 391-400.
[18] Zlotkin, E., Annu. Rev. Entomol. 1999; 44: 429-455.
[19] White, J., Med J. Aust. 1999; 171: 98.
[20] Platnick, N. I., Bull. Am. Mus. Nat. Hist. 2000; 245: 1-330.
[21] Kuhn-Nentwig, L., Schaller, J., Nentwig, W., Toxcon. 1994; 32: 287-302.
[22] Horni, A., Weickmann, D., Hesse, M., Toxicon. 2001; 39: 425-428.
[23] Isbister, G., Gray, M., Med J. Aust. 2000; 172: 303.
[24] Isbister, G., Gray, M., Proc. Aust. Soc. Clin. Exp. Pharmacol. Toxicol. 8, 15.
[25] Alexander, S. P. H., Peters, J. A., Trends Pharmacol. Sci. 1999; (Suppl.).
[26] Balaji, R. A., Sasaki, T., Gopalakrshnakone, P., Santo, K., Kini, R. M., Huat, ,B. B., Proceeding of the 5th Asia-Pacfic Congress on Animal, Plant and Aicrobial Toxins 1999; 3: 232.
[27] Reis, H. J., Prodo, M. A., Kalapothadis, E., Cordeiro, M. N. ,Qiniccrm, Q. E., Marco, L. A., Gomez, M. V., Rommane, S. M., Blochem. J. 1999; 15: 343 Pt 2: 423-8.
[28] Fletcher, J. J., Smith, R. O., Qenoghue, S. I., Nilges, M., Conner, M., Howdenm, E. H., Christie, M. J., King, C. F., Nat. Struct. Biol. 1997; 4: 559-566.
[29] Matsushita, M., Susuml, K. T., Hatakeyama, H. I., Toki, T., Miyashitta, M., Tetrahedron Letters. 1995; 36(29): 5231-5234.
[30] 黄仁槐，梁宋平．，《蜘蛛多肽神经毒素研究进展》生命科学研究．2000：4(2)：102—110
[31] 梁宋平，潘欣．，《蜘蛛肽类神经毒素研究进展》生物化学与生物物理学报．1994：21(390-396)．
[32] Brown, M. R., Sheumack, D. D., Tyler, M. I., Howden, M. H., Biochem. J 1998; 250: 401-405.
[33] Nicholson, G. M., Walsh, R., Little, M. J., Tyler, M. I., Pflugers Arch. 1998: 436: 117-126.
[34] Diochot, S., Drici, M. D., Moinier, D., Fink, M., Lazdunski, M., Br. J. Pharmacol. 1999; 126: 251-263.
[35] Grinshin, E. V., Black widow spider toxins: The present and the future. Toxicon. 1998; 36: 1693-1701.
[36] Grinshin, E. V., Polypeptide neurotoxins from the spider venoms. Eur. J. Biochem. 1999; 264: 276-280.
[37] LEIGHTON PRITCHARD AND MARK J. DUFTON. Evolutionary Trace Analysis of the Kunitz/BPTI Family of Proteins: Functional Divergence May Have been based on conformational adjustment. J. MOL. Biol. (1999) 285, 1589-1607
[38] HAGIHARA Y, SHIRAKI K, NAKAMURA T, UEGAKI K, TAKAGI M, IMANAKA T, YUMOTO N. Screening for stable mutants with amino acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI). J Biol Chem 2002 Dec 27; 277(52): 51043-8
[39] CARLACCI L. Prediction of a 12-residue loop in bovine pancreatic trypsin inhibitor: effects of buried water. Biopolymers 2001 58(4): 359-73
[40] ANTUCH, W. BERNDT, K. D. CHAVEZ, M. DELFIN, J. The NMR solution structure of Kunitz-type proteinase inhibitors from the sea anemone Anemonia stichodactyle helianthus. Eur. J. biochem. 1993. 212, 675-684
[41] CHINPAN CHEN, CHUN-HUA HSU, NING-YUAN SU. Solution structure of
a Kunitz-type Chymotrypsin inhibitor isolated from the Elapid snake bungarus fasciatus. J Biol Chem. 2001,
[42] SMITH, L. A. REID, P. F. WANG, F. C. PARCEJ, D. N., SCHMIDT, JJ, OLSON, M. A, AN D DOLLY, J. O. (1997) Biochemistry 36, 7690-7696
[43] VELARDE G, FORD RC, ROSENBERG MF, POWIS SJ. Three-dimensional structure of transporter associated with antigen processing (TAP) obtained by single Particle image analysis. J Biol Chem 2001; 276(49): 46054-63
[44] CHINPAN CHEN, CHUN-HUA HSU, NING-YUAN SU. Solution structure of a Kunitz-type Chymotrypsin inhibitor isolated from the Elapid snake bungarus fasciatus. J Biol Chem, 2001,
[45] KROWARSCH D, OTLEWSKI J. Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability. Protein Sci, 2001, 10(4): 715-24
[46] GRZESIAK A, HELLAND R, SMALAS AO, KROWARSCH D, DADLEZ M, OTLEWSKI J. Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases. J Mol Biol, 2000, 301(1): 205-17
[47] KROkOSZ KROWARSCH D, DADLEZ M, BUCZEK O, YNSKA I, SMALAS AO, OTLEWSKI J. Interscaffolding additivity: binding of P1 variants of bovine pancreatic trypsin inhibitor to four serine proteases. J Mol Biol, 1999, 289(1): 175-86
[48] CZAPINSKA H, OTLEWSKI J, KRZYWDA S, SHELDRICK GM, JASKOLSKI M. High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence. J Mol Biol, 2000, 295(5): 1237-49
[49] HELLAND R, OTLEWSKI J, SUNDHEIM O, DADLEZ M, SMALAS AO. The crystal structures of the complexes between bovine beta-trypsin and ten P1 variants of BPTI. J Mol Biol, 1999, 287(5): 923-42
[50] TAKAHASHI T. Significant role of electrostatic interactions for stabilization of protein assemblies. Adv Biophys 1997; 34: 41-54
[51] OTLEWSKI J, JASKOLSKI M, BUCZEK O, CIERPICKI T, CZAPINSKA H, KROWARSCH D, SMALAS AO, STACHOWIAK O, SZPINETA A, OAOLEZ M.
Structure-function relationship of serine protease-protein inhibitor interaction. Acta Blochim Pol, 2001; 48(2): 419-28
[52] KURODA Y, KIM PS. Folding of bovine pancreatic trypsin inhibitor (BPTI) variants in which almost half the residues are alanine. J Mol Biol, 2000, 298(3): 493-501
[53] GRZESIAK A, KROKOSZYNSKA I, KROWARSCH D, BUCZEK O, DADLEZ M, OTLEWSKI J. Inhibition of six serine proteinases of the human coagulation system by mutants of bovine pancreatic trypsin inhibitor. J Biol Chem, 2000, 275(43): 33346-52
[54] GRZESIAK A, BUCZEK O, PETRY I, SZEWCZUK Z, OTLEWSKI J. Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants. Biochim Biophys Acta, 2000, 1478
[55] CIERPICKI T, OTLEWSKI J. Determination of a high precision structure of a novel protein, Linumusitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks. : J Mol Biol, 2000, 302(5): 1179-92
[56] CIERPICKI T, BANIA J, OTLEWSKI J. NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors. : Protein Sci 2000, 9(5): 976-84
[57] 王家福，2．我国南方捕鸟蛛一新种。湖南师范大学自然科学学报，3．1993，4．16(1)：60—63
[58] Liang SP, Chen XD, Shu O, Zhang Y. The presynaptic activity of huwentoxin-Ⅰ, a neurotoxin from the Chinese bird selenocosmia huwena. Toxin. 1993, 31: 969-978
[59] Peng K, Shu Q, Liu Z, Liang S. Function and solution structure of huwentoxin-Ⅳ, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena. J Biol Chem. 2002, 277(49): 47564-71.
[60] 梁宋平，8．林莉。虎纹捕鸟蛛凝集素—Ⅰ的细胞凝集活性分析，9．中国生物化学与分子生物学报，10．2000，11．16(1)：92—95
[61] Otlewski J, Jaskolski M, Buczek O, et al. Structure-function
relationship of serine protease-protein inhibitor interaction. Acta Biochim Pol, 2001; 48(2): 419-28
[62] Anette S. N. Tarantula (Eurypelma californicum) venom, a multicomponent system, Biol chem., Hoppe-seyley. 1989, 370: 485-498
[63] Godbole S A, Krishna T G, Bhatia C R Purification and characterization of protease inhibitors from pigeon pea seeds. J Sci, Food Agric, 1994, 64: 87-93
[64] 梁宋平，谢锦云等编著，生物化学与分子生物学实验教程，．高教出版社，
[65] Zimmermann M. Ethical guidelines for investigations of experimental pain in couscious animals. Pain, 1983, 16, 109-110
[66] 禹超邦，刘德立编著，应用酶学导论，华东师大出版社
[67] Bulaj G, Goldenberg DP. Mutational analysis of hydrogen bonding residues in the BPTI folding pathway. J. Mol. Biol, 2001, 313(3): 639-56
[68] Otlewski J Jaskolski M, Buczek OStructure-function relationship of serine protease-protein inhibitor interaction. Biol Chem, Hoppe-seyley, 1989, 370: 485-498
[69] Grzesiak A, Helland R, Somalis AO, Krowarsch D, Dadlez M, Otlewski J. Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases J Mol Boil, 2000, 301(1): 205-17
[70] Capasso C, Rizzi, M. Crystal structure of the bovine alpha-chymotrypsin: Kunitz inhibitor complex: An example of multiple protein: protein recognition sites. J Mol Recognit 1997, 10(1): 26-35
[71] Chen C, Hsu CH, Su NY, Lin YC, Chiou SH, Wu SH. Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus. J Biol Chem, 2001, 276(48): 45079-87
[72] Nishio H, Katoh E, Yamazaki T, Inui T, Nishiuchi Y. Structure-activity relationships of calcicludine and dendrotoxin-Ⅰ, homologous peptides acting on different targets, calcium and potassium channels. Biochem Biophys Res commun 1999, 262(2): 319-21
[73] Schweitz H, Bruhn T, Guillemare E, Moinier D, Kalicludines and kaliseptine. Two different classes of sea anemone toxins for voltage sensitive K+ channels. J Biol Chem, 1995, 270(42): 25121-6.