蜘蛛肽类毒素的结构与功能研究
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摘要
1：虎纹捕鸟蛛(Selenocosmia huwena)是在我国西南地区发现的一蜘蛛新种，属于捕鸟蛛科(Theraphosidae)。该蛛穴居地下，个体大，毒性强。其粗毒中含有多种成分。我们应用离子交换和反相高效液相色谱已经从中分离出多种神经毒素如(HWTX-Ⅰ，HWTX-Ⅱ，HWTX-Ⅲ，HWTX-Ⅳ，SHL-1)，并对这些毒素进行鉴定和研究。
本文报道从虎纹捕鸟蛛(Selenocoima huwena)粗毒中，结合阳离子交换和反相高效液相色谱分离纯化出一种昆虫毒素，命名为虎纹捕鸟蛛毒素-Ⅴ(Huwentoxin-Ⅴ，HWTX-Ⅴ)；其天然突变体(the mutant of Huwentoxin，mHWTX-Ⅴ)也同时纯化出来。经MALDI-TOF质谱分析，HWTX-Ⅴ的相对分子量为4111.42±0.4Da，mHWTX-Ⅴ的分子量是3877.08±0.4Da。经DTT还原，进一步用碘乙酰胺修饰，它们的分子量分别增加到4460.2Da和4225.1Da。因此我们可以确定HWTX-Ⅴ和mHWTX-Ⅴ都有三对二硫键。从它们的实测分子量和氨基酸序列计算分子量都相差6Da，进一步证实了它们的6个半胱氨酸都形成了3对二硫键。利用Edman降解气相蛋白质自动测序仪得到Huwentoxin-Ⅴ为一单一多肽链，含35个氨基酸，其中6个半胱氨酸残基形成3对二硫键，其氨基酸全序列为NH2-ECRWYLGGCSQDGDCCKHLQCHSNYEWCVWDGTFSCOOH；同样mHuwentoxin-Ⅴ也为单一多肽链，含有33个的氨基酸，其氨基酸序列为NH2-ECRWYLGGCSQDGDCCKHLQCHSNYEWCVWDGT-COOH。从氨基酸序列上看，mHuwentoxin-Ⅴ只在C末端比Huwentoxin-Ⅴ少Phe和Ser残基，它们的结构有很高的相似性。根据HWTX-Ⅴ的cDNA分析，我们认为这两个毒素来自于同一个基因，是经过翻译后加工形成的天然突变体。
HWTX-Ⅴ通过腹腔注射， 能使蝗虫和蜚蠊产生可逆的剂量依赖性的麻痹作用，麻痹时间长达十多个小时，在高剂量(100μg／g)时可使蝗虫和蜚蠊致死，对蝗虫的ED_(50)=16±5μg／g。而HWTX-Ⅴ腹腔注射(200μ／g)和脑室注射(30μg／g)对小鼠没有异常反应，也不能阻断膈神经膈肌标本的神经肌肉传递，可以得出HWTX-Ⅴ是一种对昆虫有特异性作用的昆虫毒素，对哺乳动物没有作用。但是
湖一{，J Ij.}.范人·，护2(}(l〕}.11 1.公!l‘}、业l众义
mHTWX一V对蝗虫、蜚蜿及小鼠都没有明显反应。它们的结构一与，)J能
的关系、:J‘以推断出Ilw，l.x一v的e末端的I，l飞e”‘不一1 5 er’“几为一lwl、x一v的关
键性残基，决定着HwTX一v的昆虫活性。
利用TCEP部分还原测序法、结合酶解方法确定了HWXT一V的二
硫键的位置，它们是eysZ一eys16，eySg一eys21，eys15一Cys28即以
1一4，2一5，3一6的方式连接。经同源性搜索，发现这两种毒素与多
种蜘蛛毒素具有较高的同源性，如Thrixopelmo prurien:中的
proTx一I(65%)，Scodra grise切es中的SGTx一l(57%)，Hysreroerares
giga:中的sNx一452(55%)，以及Grammostolas尸atulara中的
HaTXI(5470)/HaTxZ(54%)。从这些同源序列的二硫键的结合方式看
来，HwTX一v和mHwTX一v很可能属(1 nhibitory Cysteine Knot)ICK
结构模体。
The Chinese bird spider Selenocomia huwena is one of the most venomous spiders in China, which distributes in southwestern hilly area of China and habitually lives in the holes underground. The venom of this spider ontains a mixture of compounds with different types of biological activity. Many kinds of neurotoxic toxins such as HWTX-I, HWTX-II, HWTX-III, and HWTX-IV etc have been isolated by combination of ion exchange chromatography and reverse phase high performance liquid chromatography.
A neurotoxic peptide (named huwentoxin-V) was purified from the venom of the spider by a combination of ion exchange chromatography and reverse phase HPLC. HWTX-V has 35 amino acid residues, with the molecular mass 4111.4Da. The amino acid sequence has been determined as NH2-ECRWYLGGCSQDGDCCKHLQCHSN-YEWCVWDGTFS-COOH, which consists of 6 Cys, formed three pairs of disulfide bridges. A natural mutant of the toxin (called mHuwentoxin-V) was also isolated from the venom. The complete amino acid sequence is NH2.ECRWYLGGCSQDGDCCKHLQCHSNYE -WCVWDGT-COOH determined by automated Edman degradation. mHWTX-V was only truncated two amino acid residues from the C-terminus of HWTX-V, and its molecular weight is 3877.1 Da determined by mass spectrometry. There are three disulfide bridges in each of two peptides demonstrated by mass spectrometry. According to the cDNA of HWTX-V, HWTX-V and mHWTX-V might be come from the same gene. The difference of two toxins may be result from post-translation modification.
HWTX-V can reversibly paralyze locusts and cockroaches for several hours with an ED50 value as 16+5ug/g to locusts, and larger
dose of the toxin can cause death. The neurotoxic symptoms induced
by intra-abdominal injection of Huwentoxin-V were paralysis, immobilization and last gradually recovery. HWTX-V was not shown to block neuromuscular transmission in isolated mouse phrenic nerve diaphragm preparation. HWTX-V also had not evidently effect on mice by intra-abdominal injection with high dose of 400ug/g. We can conclude that Huwentoxin-V is a potent insecticidal toxin, without no verbrate toxicity. However, mHWTX-V shows no significant effect on locusts and cockroaches. The structure-activity relationship indicates that the residues Phe34 and Ser35 in the C-terminus of HWTX-V are the key residues of the biological activity.
To assign the disulfide bonds of HWTX-V, we employ the method of partial reduction using TCEP. The labeled thiols were identified by Edman degradation. The results indicated specific location of three disulfide bonds. The three-disulfide linkage of HWTX-V has been assigned as Cys2-Cysl6, Cys9-Cys21, and Cysl5-Cys28. The primary
structure of two toxins exhibited sequence identity with other spider
toxins such as ProTx-I (65%), a toxin from the venom the tarantula spider Thrixopelma pruriens, SGTx (57%), a toxin from the spider Scodra grisepes, SNX-482 (55%), a toxin from the tarantula spider Hysterocrates gigas and HaTxl (54%) /HaTx2 (54%) from the spider Grammostola spatulata. According to the sequences similarity and the same disulfide linkage of HWTX-V family with other toxins with known 3D structures, we can postulate that HWTX-V may belong to ICK (Inhibitory Cysteine Knot) motif.
本文报道从虎纹捕鸟蛛(Selenocoima huwena)粗毒中，结合阳离子交换和反相高效液相色谱分离纯化出一种昆虫毒素，命名为虎纹捕鸟蛛毒素-Ⅴ(Huwentoxin-Ⅴ，HWTX-Ⅴ)；其天然突变体(the mutant of Huwentoxin，mHWTX-Ⅴ)也同时纯化出来。经MALDI-TOF质谱分析，HWTX-Ⅴ的相对分子量为4111.42±0.4Da，mHWTX-Ⅴ的分子量是3877.08±0.4Da。经DTT还原，进一步用碘乙酰胺修饰，它们的分子量分别增加到4460.2Da和4225.1Da。因此我们可以确定HWTX-Ⅴ和mHWTX-Ⅴ都有三对二硫键。从它们的实测分子量和氨基酸序列计算分子量都相差6Da，进一步证实了它们的6个半胱氨酸都形成了3对二硫键。利用Edman降解气相蛋白质自动测序仪得到Huwentoxin-Ⅴ为一单一多肽链，含35个氨基酸，其中6个半胱氨酸残基形成3对二硫键，其氨基酸全序列为NH2-ECRWYLGGCSQDGDCCKHLQCHSNYEWCVWDGTFSCOOH；同样mHuwentoxin-Ⅴ也为单一多肽链，含有33个的氨基酸，其氨基酸序列为NH2-ECRWYLGGCSQDGDCCKHLQCHSNYEWCVWDGT-COOH。从氨基酸序列上看，mHuwentoxin-Ⅴ只在C末端比Huwentoxin-Ⅴ少Phe和Ser残基，它们的结构有很高的相似性。根据HWTX-Ⅴ的cDNA分析，我们认为这两个毒素来自于同一个基因，是经过翻译后加工形成的天然突变体。
HWTX-Ⅴ通过腹腔注射， 能使蝗虫和蜚蠊产生可逆的剂量依赖性的麻痹作用，麻痹时间长达十多个小时，在高剂量(100μg／g)时可使蝗虫和蜚蠊致死，对蝗虫的ED_(50)=16±5μg／g。而HWTX-Ⅴ腹腔注射(200μ／g)和脑室注射(30μg／g)对小鼠没有异常反应，也不能阻断膈神经膈肌标本的神经肌肉传递，可以得出HWTX-Ⅴ是一种对昆虫有特异性作用的昆虫毒素，对哺乳动物没有作用。但是
湖一{，J Ij.}.范人·，护2(}(l〕}.11 1.公!l‘}、业l众义
mHTWX一V对蝗虫、蜚蜿及小鼠都没有明显反应。它们的结构一与，)J能
的关系、:J‘以推断出Ilw，l.x一v的e末端的I，l飞e”‘不一1 5 er’“几为一lwl、x一v的关
键性残基，决定着HwTX一v的昆虫活性。
利用TCEP部分还原测序法、结合酶解方法确定了HWXT一V的二
硫键的位置，它们是eysZ一eys16，eySg一eys21，eys15一Cys28即以
1一4，2一5，3一6的方式连接。经同源性搜索，发现这两种毒素与多
种蜘蛛毒素具有较高的同源性，如Thrixopelmo prurien:中的
proTx一I(65%)，Scodra grise切es中的SGTx一l(57%)，Hysreroerares
giga:中的sNx一452(55%)，以及Grammostolas尸atulara中的
HaTXI(5470)/HaTxZ(54%)。从这些同源序列的二硫键的结合方式看
来，HwTX一v和mHwTX一v很可能属(1 nhibitory Cysteine Knot)ICK
结构模体。
The Chinese bird spider Selenocomia huwena is one of the most venomous spiders in China, which distributes in southwestern hilly area of China and habitually lives in the holes underground. The venom of this spider ontains a mixture of compounds with different types of biological activity. Many kinds of neurotoxic toxins such as HWTX-I, HWTX-II, HWTX-III, and HWTX-IV etc have been isolated by combination of ion exchange chromatography and reverse phase high performance liquid chromatography.
A neurotoxic peptide (named huwentoxin-V) was purified from the venom of the spider by a combination of ion exchange chromatography and reverse phase HPLC. HWTX-V has 35 amino acid residues, with the molecular mass 4111.4Da. The amino acid sequence has been determined as NH2-ECRWYLGGCSQDGDCCKHLQCHSN-YEWCVWDGTFS-COOH, which consists of 6 Cys, formed three pairs of disulfide bridges. A natural mutant of the toxin (called mHuwentoxin-V) was also isolated from the venom. The complete amino acid sequence is NH2.ECRWYLGGCSQDGDCCKHLQCHSNYE -WCVWDGT-COOH determined by automated Edman degradation. mHWTX-V was only truncated two amino acid residues from the C-terminus of HWTX-V, and its molecular weight is 3877.1 Da determined by mass spectrometry. There are three disulfide bridges in each of two peptides demonstrated by mass spectrometry. According to the cDNA of HWTX-V, HWTX-V and mHWTX-V might be come from the same gene. The difference of two toxins may be result from post-translation modification.
HWTX-V can reversibly paralyze locusts and cockroaches for several hours with an ED50 value as 16+5ug/g to locusts, and larger
dose of the toxin can cause death. The neurotoxic symptoms induced
by intra-abdominal injection of Huwentoxin-V were paralysis, immobilization and last gradually recovery. HWTX-V was not shown to block neuromuscular transmission in isolated mouse phrenic nerve diaphragm preparation. HWTX-V also had not evidently effect on mice by intra-abdominal injection with high dose of 400ug/g. We can conclude that Huwentoxin-V is a potent insecticidal toxin, without no verbrate toxicity. However, mHWTX-V shows no significant effect on locusts and cockroaches. The structure-activity relationship indicates that the residues Phe34 and Ser35 in the C-terminus of HWTX-V are the key residues of the biological activity.
To assign the disulfide bonds of HWTX-V, we employ the method of partial reduction using TCEP. The labeled thiols were identified by Edman degradation. The results indicated specific location of three disulfide bonds. The three-disulfide linkage of HWTX-V has been assigned as Cys2-Cysl6, Cys9-Cys21, and Cysl5-Cys28. The primary
structure of two toxins exhibited sequence identity with other spider
toxins such as ProTx-I (65%), a toxin from the venom the tarantula spider Thrixopelma pruriens, SGTx (57%), a toxin from the spider Scodra grisepes, SNX-482 (55%), a toxin from the tarantula spider Hysterocrates gigas and HaTxl (54%) /HaTx2 (54%) from the spider Grammostola spatulata. According to the sequences similarity and the same disulfide linkage of HWTX-V family with other toxins with known 3D structures, we can postulate that HWTX-V may belong to ICK (Inhibitory Cysteine Knot) motif.
引文
1. Scott, , R. H., Sutton, K.G., Annette, C. D. Trends in Neuroseiencees. 1993; 16(4): 153-159.
2. Grinshin, E. Eur. J. Biochem. 1999; 264: 276-280.
3. Longenecker, H. E., Hurbut, W. P., Mauro, A., et al. Nature. 1970; 225: 701.
4. Frontail, N., Ceccarelli, B., Gorio, A., et al. J. Cell Bio. 1976; 68: 462.
5. Hulbut, W. P., Lezzi, N., Fesce, R., Ceccarelli, B. J. Physiol. 1990; 425: 501-526.
6. Petrenko, A. G., Kovalenko, V. A. Shamotienk0, Z. N., et al. EMBO Joural. 1990; 9: 2023.
7. Kiyatkin, N. I., Dulubova, E., Cheknovskaya, I. I., et al. FEBS Letters. 1990; 270: 127.
8. Lurnev, A. V., Qemin, V. V., et al. Bioong Khim. 1991; 17: 1021-26.
9. Erishin, E. V. Toxicon; 1998; 36(11): 1693-1701.
10. Michaely, P., Bennett, V. Trends Cell Biol. 1992; 2:127-129.
11. Danilevich, V. N., Luk'ianov, S. A.,Grishin, E. V. Bioorg Khim. 1999; 25(7): 537-547.
12. Sheumack, D. D., Classens, R. Whiteleg, N. M., et al. FEBS Letters. 1985; 181: 154.
13. Brown, M. R., Sheumack, D. D., Tyler, M. I., et al. Biochem. J. 1988; 250" 401.
14. Nicholson, G. M., Little, M. J., Tyler, M., Narahashi, T. Toxicon. 1996; 34(11-I2):1443-53.
15. Skinner, W. S., Adams, M. E., Quistad, X. et al.J. Biol. Chem. 1989; 264:2150.
16. Admas, M E., Binokas, V. P., Hasewage, L., Veneva, V. J. J.
Biol. Chem. 1990; 265: 861-867.
17. Ertel, E. A.,Waren, V. J., Adams, M. E., et al., Biochemstry. 1994; 33:5098-5108.
18. Mintz, I. M., Venema, V. J., Swiderek, K. M., Nature. 1992; 355:827-829.
19. Fletcher, J. I., Smith, R. O., Donoghue, S. I., Nilges, M., Connor, M., et al., Nat. Struct. Bio. 1997; 4:559-566.
20. Little, M. J., Wilson, H., Zappia, C., Cestle, S., et al., FEBS Letter. 1998; 439(3):246-52.
21. Rash, L. D., Hodgson, W. C., Toxicon. 2002; 40:225-254.
22. Escoubas, P., Diochot, S., Gerardo, C., Biochimie. 2000; 82:893-907.
23. Young, A. R., Pincus, S. J., Toxicon. 2001; 39:391-400.
24. Zlotkin, E., Annu. Rev. Entomol. 1999; 44:429-455.
25. White, J., Med. J. Aust. 1999; 171:98.
26. Platnick, N. I., Bull. Am. Mus. Nat. Hist. 2000; 245:1-330.
27. Kuhn-Nentwig, L., Schaller, J., Nentwig, W., Toxcon. 1994; 32:287-302.
28. Horni, A., Weickmann, D., Hesse, M., Toxicon. 2001; 39:425-428.
29. Isbister, G., Gray, M., Med. J. Aust. 2000; 172:303.
30. Isbister, G., Gray, M., Proc. Aust. Soc. Clin. Exp. Pharmacol. Toxicol. 8,15.
31. Alexander, S. P. H., Peters, J. A., Trends PharmacoI. Sci. 1999; (Suppl.).
32. Balaji, R. A., Sasaki, Y., 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.
33. Reis, H. J., Prodo, M. A., Kalapothadis, E., Cordeiro, M. N. ,Qiniccrm, Q. E.,Marco, L. A., Gomez, M. V., Rommane, S. M., Biochem. J. 1999; 15:343 Pt 2:423-8.
34. 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.
35. Matsushita, M., Susuml, K. T., Hatakeyama, H. I., Toki, T., Miyashitta, M., Tetrahedron Letters. 1995; 36(29):5231-5234.
36．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
37．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(390-395)．
38. Brown, M. R., Sheumack, D. D.,Tyler, M.I.,Howden, M.H., Biochem. J. 1998; 250:401-405.
39. Nicholson, G. M., Walsh, R., Little, M. J., Tyler, M. I., Pflugers Arch. 1993; 436:117-126.
40. Diochot, S., Drici, M. D., Moinier, D., Fink, M., Lazdunski, M., Br. J. Pharmacol. 1999; 126:251-263.
2. Grinshin, E. Eur. J. Biochem. 1999; 264: 276-280.
3. Longenecker, H. E., Hurbut, W. P., Mauro, A., et al. Nature. 1970; 225: 701.
4. Frontail, N., Ceccarelli, B., Gorio, A., et al. J. Cell Bio. 1976; 68: 462.
5. Hulbut, W. P., Lezzi, N., Fesce, R., Ceccarelli, B. J. Physiol. 1990; 425: 501-526.
6. Petrenko, A. G., Kovalenko, V. A. Shamotienk0, Z. N., et al. EMBO Joural. 1990; 9: 2023.
7. Kiyatkin, N. I., Dulubova, E., Cheknovskaya, I. I., et al. FEBS Letters. 1990; 270: 127.
8. Lurnev, A. V., Qemin, V. V., et al. Bioong Khim. 1991; 17: 1021-26.
9. Erishin, E. V. Toxicon; 1998; 36(11): 1693-1701.
10. Michaely, P., Bennett, V. Trends Cell Biol. 1992; 2:127-129.
11. Danilevich, V. N., Luk'ianov, S. A.,Grishin, E. V. Bioorg Khim. 1999; 25(7): 537-547.
12. Sheumack, D. D., Classens, R. Whiteleg, N. M., et al. FEBS Letters. 1985; 181: 154.
13. Brown, M. R., Sheumack, D. D., Tyler, M. I., et al. Biochem. J. 1988; 250" 401.
14. Nicholson, G. M., Little, M. J., Tyler, M., Narahashi, T. Toxicon. 1996; 34(11-I2):1443-53.
15. Skinner, W. S., Adams, M. E., Quistad, X. et al.J. Biol. Chem. 1989; 264:2150.
16. Admas, M E., Binokas, V. P., Hasewage, L., Veneva, V. J. J.
Biol. Chem. 1990; 265: 861-867.
17. Ertel, E. A.,Waren, V. J., Adams, M. E., et al., Biochemstry. 1994; 33:5098-5108.
18. Mintz, I. M., Venema, V. J., Swiderek, K. M., Nature. 1992; 355:827-829.
19. Fletcher, J. I., Smith, R. O., Donoghue, S. I., Nilges, M., Connor, M., et al., Nat. Struct. Bio. 1997; 4:559-566.
20. Little, M. J., Wilson, H., Zappia, C., Cestle, S., et al., FEBS Letter. 1998; 439(3):246-52.
21. Rash, L. D., Hodgson, W. C., Toxicon. 2002; 40:225-254.
22. Escoubas, P., Diochot, S., Gerardo, C., Biochimie. 2000; 82:893-907.
23. Young, A. R., Pincus, S. J., Toxicon. 2001; 39:391-400.
24. Zlotkin, E., Annu. Rev. Entomol. 1999; 44:429-455.
25. White, J., Med. J. Aust. 1999; 171:98.
26. Platnick, N. I., Bull. Am. Mus. Nat. Hist. 2000; 245:1-330.
27. Kuhn-Nentwig, L., Schaller, J., Nentwig, W., Toxcon. 1994; 32:287-302.
28. Horni, A., Weickmann, D., Hesse, M., Toxicon. 2001; 39:425-428.
29. Isbister, G., Gray, M., Med. J. Aust. 2000; 172:303.
30. Isbister, G., Gray, M., Proc. Aust. Soc. Clin. Exp. Pharmacol. Toxicol. 8,15.
31. Alexander, S. P. H., Peters, J. A., Trends PharmacoI. Sci. 1999; (Suppl.).
32. Balaji, R. A., Sasaki, Y., 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.
33. Reis, H. J., Prodo, M. A., Kalapothadis, E., Cordeiro, M. N. ,Qiniccrm, Q. E.,Marco, L. A., Gomez, M. V., Rommane, S. M., Biochem. J. 1999; 15:343 Pt 2:423-8.
34. 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.
35. Matsushita, M., Susuml, K. T., Hatakeyama, H. I., Toki, T., Miyashitta, M., Tetrahedron Letters. 1995; 36(29):5231-5234.
36．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
37．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(390-395)．
38. Brown, M. R., Sheumack, D. D.,Tyler, M.I.,Howden, M.H., Biochem. J. 1998; 250:401-405.
39. Nicholson, G. M., Walsh, R., Little, M. J., Tyler, M. I., Pflugers Arch. 1993; 436:117-126.
40. Diochot, S., Drici, M. D., Moinier, D., Fink, M., Lazdunski, M., Br. J. Pharmacol. 1999; 126:251-263.