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研究生:朱苑萍
研究生(外文):Yuan-Ping Chu
論文名稱:台灣雨傘節beta-Bungarotoxins:異構毒素、作用標的與基因結構之研究
論文名稱(外文):Taiwan Banded Krait beta-Bungarotoxins:Novel Isotoxins, Targeting and Gene Organization
指導教授:張榮賢
指導教授(外文):Long-Sen Chang
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:105
中文關鍵詞:台灣雨傘節基因異構毒素
外文關鍵詞:A chainexon skippingbeta-BungarotoxinisotoxinBungarus multicinctus
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  • 被引用被引用:3
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beta-Bungarotoxin純化自台灣雨傘節(學名Bungarus multicinctus,俗名Taiwan banded krait)蛇毒,由A chain與B chain兩個Subunits以雙硫鍵鍵結組成。 本實驗經由離子交換樹脂及高壓液相層析儀自雨傘節蛇毒中純化出BM12與BM13兩個新的beta-Bgt isotoxins,並利用質譜分析及N-termianl sequencing和peptide mapping得知BM12與BM13可能由相同A chian但不同B chain所組成。 將BM12及BM13進行抗原性、Phospholipase A2活性、二級結構及螢光分析,顯示可能由於B chain影響使得BM13抗原性大於BM12,同時也使得BM13 PLA2活性較BM12高,在與二價離子反應上BM12與BM13之酵素活性會受Sr2+、Ba2+離子所抑制,而且BM12與BM13對Mg2+、Ca2+、Sr2+、Ba2+之結合能力並不相同,可知不同A chain及B chain subunits的組合方式,會造成b-Bgt活性及結構上的差異。
Yeast-Two-Hybrid系統中,beta-Bgt B chain會和一potassium channel binding protein作用,將之轉形於E. coli系統中表現。 二級結構及螢光分析顯示,自E. coli系統表現之potassium channel binding protein具有Ca2+結合能力,證實其EF hand為具有功能之單元結構,但在In vitro cross-linking及Co-immunoprecipitation實驗中並無法顯現B chain可能和此一蛋白產生結合反應。
以PCR方式自雨傘節染色體放大出四段不同A chain genes,分別為A1 chain、A1a chain、A2 chain及A8 chain genes,四段基因之核苷酸序列同源性高達97.5%,與cDNA進行分析比對發現A1 chain之Signal peptide region有一段Intron的Insertion,使得A1 chain gene具有4個Exon及3個Intron,而A2 chain gene則使用相對於A1 chain gene鄰近的Intron 1序列與exon 2 region形成其exon 1,造成A2 chain只有3個Exon與2個Intron,也因此導致A2 chain signal peptide序列只有25個胺基酸,不同於A1 chain有27個胺基酸的Signal peptide。 然而,A1及A2 chain genes之 Promoter region序列及Transcription factor binding-sites具有極高的保留性,認為A chain genes可能由相同的機制所調控。 5’-RACE的分析指出,Bungarus multicinctus毒腺內之mRNAs中,27個胺基酸的Signal peptide 是A chain mRNAs中Pre-dominant species。 將目前已發表之Phospholipase A2 genes和cDNAs進行分析比較,證實此為首次提出蛇毒蛋白藉由Exon skipping方式產生不同的Signal peptide sequence。
beta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay.
The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins.
中文摘要------------------------------3
英文摘要------------------------------5
緒論----------------------------------6
縮寫----------------------------------17
實驗材料------------------------------18
實驗方法------------------------------20
實驗結果------------------------------34
討論----------------------------------47
圖表----------------------------------54
參考文獻------------------------------91
Abe, T., Alema, S. and Miledi, R. (1977) Isolation and characterization of presynaptically acting neurotoxins from the venom of Bungarus snakes. Eur. J. Biochem. 80, 1-12.
Asai, Y. and Watanabe, S. (1999) Effect of divalent cation on the membrane properties of the lipid A analog E5531. Drug Dev. Ind. Pharm. 25, 1107-1113.
Breeze, A. L. and Dolly, J. O. (1989) Interaction between discrete neuronal membrane binding sites for the putative K+ channel ligands beta-bungarotoxin, dendrotoxin and mast-cell-degranulating peptide. Eur. J. Biochem. 178, 771-778.
Chang, C. C. (1985) Neurotoxins with phospholipase A2 activity in snake venoms. Proceedings of the National Science Council ROC. (B) 9, 126-142.
Chang, L. S. and Yang, C. C. (1988) Role of N-terminal region of the A chain in beta1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan banded krait) venom. J. protein chem. 12, 469-475.
Chang, L. S. and Yang, C. C. (1993) Separation and characterization of the A chain and B chain in beta1-bungarotoxin from Bungarus multicinctus (Taiwan banded krait) venom. J. Protein Chem. 12, 496-475.
Chang, L. S., Wen, E. Y. and Chang, C. C. (1996) The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2. J. Protein Chem. 15, 255-260.
Chang, L. S., Lin, S. k., Huang, S. B. and Hsiao, M. (1999) Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mRNAs. Nucleic Acid Res. 27, 3970-3975.
Chapell, R. and Rosenberg, P. (1992) Specificity of action of beta-bungarotoxin on neurotransmitter release and its inhibitory effects on acetylcholine release from synaptosomes. Toxicon 30, 621-633.
Chu, S. T. and Chen, Y. H. (1991) Role of the N-terminal region of phospholipase A2 subunit of b1-bungarotoxin in the toxin-Ca2+ complex-formation. Biochem. J. 278, 481-486.
Chu C. C., Chu, S. T., Chen, S. W. and Chen, Y. H. (1994) The non-phospholipase A2 subunit of beta-bungarotoxin plays an important role in the phospholipase A2-independent neurotoxic effect: characterization of three isotoxins with a common phospholipase A2 subunit. Biochem. J. 303, 171-176.
Čopic A., Vučemilo, N., Gubenšek, F. and Križaj, I. (1999) Identificaion and purification of a novel receptor for secretory phospholipase A2 in porcine cerebral cortex. J. Biol. Chem. 274, 26315-26320.
Danishefsky, A. T., Housset, D., Kim, K. S., Tao, F., Fuchs, J., Woodard, C. and Wlodawer, A. (1993) Crevice-forming mutants in the rigid core of bovine pancreatic trypsin inhibitor: crystal structures of F22A, Y23A, N43G, and F45A. Protein Sci. 2, 577-587.
Deshimaru, M., Ogawa, T., Nakashima, K., Nobuhisa, i., Chijwa, T., Shimohigashi, Y., Fukumaki, Y., Niwa, M., Yamashina, I., Hattori, S. and Ohno, M. (1996) Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS lett. 397, 83-88.
Dodds, D. C., Omeis, I. A., Cushman, S. J., Helms, J. A. and Perin, M. S. (1997) Neuronal pentraxin receptor, a novel putative integral membrane pentrxin that interacts with neuronal pentraxin 1 and 2 and taipoxin-associated calcium-binding protein 49. J. Biol. Chem. 268, 699-705.
Dyer, F. and Penner, R. (1987) The actions of presynaptic snake toxins on membrane currents of motor nerve terminals. J. Physiol. Lond. 386, 455-463.
Herkert, M., Shakhman, O., Schweins, E. and Becker, C. M. (2001) beta-Bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization. Eur. J. Neurosci. 14, 821-828.
Hseu, M. J., Yen, C. H. and Tzeng, M. C. (1999) Crocalbin: a new calcium-binding protein that is also a binding protein for crotoxin, a neurotoxic phospholipase A2. FEBS lett. 445, 440-444.
Jeyasslan, K., Armugam, A., Donghui, M. and Tan, N. H. (2000) Structure and phylogeny of the venom group I phospholipase A2 gene. Mol. Biol. Evol. 17, 1010-1021.
Kelly, R. B., Von Wedel, R. J. and Strong, P. N. (1979) Phospholipase-dependent and phospholipase-independent inhibition of transmitter release by beta-bungarotoxin. Adv. Cytopharmac. 3, 77-85.
Kondo, K., Toda, H., Narita, K. and Lee, C. Y. (1982a) Amino acid sequence of beta2-Bungarotoxin from Bungarus multicinctus venom. The amino acid substitution in the B chains. J. Biochem. 91, 1519-1530.
Kondo, K., Toda, H., Narita, K. and Lee, C. Y. (1982b) Amino acid sequence of three beta-bungarotoxins (beta3-, beta4-, and beta5-bungaorotixns) from Bungarus venom. Amino acid substitutions in the A chains. J. Biochem. 91, 1531-1548.
Kordiš, D. and Gubenšek, F. (1996) Ammodytoxin C gene helps to elucidate the irregular structure of Crotalinase group II phospholipase A2 genes. Eur. J. Biochem. 240, 83-90.
Križaj, I. and Gubenšek, F. (2000) Neuronal receptors for phospholipase A2 and beta-neurotoxicity. Biochimie 82, 807-817.
Kwong, P. D., McDonald, N. Q., Sigler, P. B. and Hendrickson, W. A. (1995) Structure of beta2-bungarotoxin: potassium channel binding by Kunitz modules and targeted phospholipase action. Structure 3, 1109-1119.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
Lin, W. Z., Chu, S. T. and Chen, T. H. (1984) Optical activity and conformation of beta-bungarotoxin in solution. Proc. Natl. Sci. Counc. ROC. 8, 113-118.
Montecucco, C. and Rossetto, O. (2000) How do presynaptic PLA2 neurotoxins block nerve terminals? TIBS 25, 266-270.
Ohno, M., Menez, R., Ogawa, T., Dansa, J. M., Shimohigashi, Y., Fromen, C., Ducancel, F., Zinn-Justin, S., Le Du, M. H., Boulain, J. C., Tamiya, T. and Menez, A. (1998) Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? Prog. Nucleic Acid Res. Mol. Biol. 59, 307-364.
Porton, B., Kao, H. T. and Greengard, P. (1999) Characterization of Trascripts from the synapsin III gene locus. J. Neurochem. 73, 2266-2271.
Rehm, H. and Betz, H. (1982) Binding of beta-bungarotoxin to synaptic membrane fractions of chick brain. J. Biol. Chem. 257, 10015-10022.
Rowan, E. G. and Harvey, A. L. (1988) Potassium channel blocking actions of beta-bungarotoxin and related toxins on mouse and frog motor nerve terminals. Br. J. Pharmacol. 94, 839-847.
Scott, D. L., Achari, A., Vidal, J. C. and Sigler, P. B. (1992) Crystallographic and biochemical studies of the inactive Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. J. Biol. Chem. 267, 22645-22657.
Skarzynski T. (1992) Crystal structure of alpha-dendrotoxin from the green mamba venom and its comparison with the structure of bovine pancreatic trypsin inhibitor. J. Mol. Biol. 224, 671-683.
Spreafico, F., and Barski, J. J. and Farina, C. (2001) Mouse DREAM/Calsenilin/KChIP3: gene structure, coding potential, and expression. Mol. Cellular Neurosci. 17, 1-16.
Šribar, J., Čopic, A., Pariš, A., Sherman, N. E., Gubenšek, F., Fox, J. W. and Križaj, I. (2001) A high affinity acceptor for phospholipase A2 with neurotoxic activity is a calmodulin. J. Biol. Chem. 276, 12493-12496.
Strong, P. N., Goerke, J., Oberg, S. G. and Kelly, R. B. (1976) beta-Bungarotoxin, a pre-synaptic toxin with enzymatic activity. Proc. Nat. Acad. Sci. USA. 73, 178-182.
Wu, P. F. and Chang, L. S. (2000) Genetic organization of A chain and B chain of beta-bungarotoxin from Taiwan banded krait (Bungarus multicinctus). Eur. J. Biochem. 267, 4668-4675.
Wu, P. F. and Chang, L. S. (2001) Expression of A chain and B chain of beta-bungarotoxin from Taiwan banded krait: the functional implication of the interchain disulfide bond between A chain and B chain. J. Protein Chem. 20, 413-420.
Yang, C. C. and Lee, H. J. (1986) Selective modification of tyrosin-68 in beta1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan banded krait). J. Protein Chem. 5, 15-28.
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