(3.239.192.241) 您好!臺灣時間:2021/03/02 19:56
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:張捷
研究生(外文):Chieh Chang
論文名稱:台灣眼鏡蛇毒5端核苷酸水解酶(V5NTD)cDNA定序與結晶結構分析
論文名稱(外文):Crystal structure analysis and cDNA sequencing of venom 5’-nucleotidase from Naja atra
指導教授:吳文桂
指導教授(外文):Wu, Wen Guey
口試委員:許素菁簡昆鎰
口試委員(外文):Hsu, Shu ChingChien, Kun Yi
口試日期:2016-12-30
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2016
畢業學年度:105
語文別:中文
論文頁數:52
中文關鍵詞:蛇毒蛋白5端核苷酸酶蛋白質結晶結構
外文關鍵詞:snake venom protein5’-nucleotidaseprotein crystal structure
相關次數:
  • 被引用被引用:0
  • 點閱點閱:73
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:9
  • 收藏至我的研究室書目清單書目收藏:0
蛇毒5端核苷酸酶(snake venom 5’-nucleotidase, V5NTD)常現於各種毒蛇的毒液之中。然而由於其在眼鏡蛇毒液內的含量相對稀少(約佔整體眼鏡蛇毒蛋白質重量之0.38%),因此過去的眼鏡蛇毒咬傷研究缺少對V5NTD生理功能的討論。近年來的研究指出,V5NTD能夠藉由水解血液中單磷酸腺苷(adenosine monophosphate, AMP)引發下游嘌呤傳訊路徑來抑制凝血作用。為了探討V5NTD代謝AMP的機制,我們建立了一套蛋白質純化機制,從中華眼鏡蛇(Naja atra)分離出V5NTD蛋白質並結晶,成功得到了1.9 Å高解析度的蛋白質結晶結構數據。透過結構上的比較,我們發現V5NTD與其在人類的同源蛋白——CD73有高度的相似,而CD73被認為是參與調控免疫反應的胞外酵素(ecto-enzyme)。在完成用cDNA定序V5NTD一級結構之後,我們利用分子置換的方式建構V5NTD的結構——V5NTD單一分子為60 kD並以靜電力相吸形成二聚體。這份研究不僅提供了從粗蛇毒內純化V5NTD的方法,也提出了第一個脊椎動物天然的5端核苷酸酶的結構,解釋了在自然界中此酵素的二聚體介面的交互作用,以及其與人類同源蛋白不同的的醣化修飾,為往後探討V5NTD於蛇毒咬傷時的生理功能提供分子結構基礎。
Venom 5’-nucleotidases (V5NTD) are widely represented in venomous snakes. Its biological function was less extensively investigated in the past cobra envenomation research owing to its scarcity in cobra venom (0.38% of Taiwan cobra venom proteins). In recent studies, it has been implied to conduct anti-coagulation by liberating of extracellular adenosine through purinergic pathways. To understand how V5NTD catalysed AMP, we purified V5NTD from Taiwan cobra (Naja atra) crude venom to determine its 3D structure at 1.9 Å by X-ray crystallography. By structural analysis, we found the crystal structure of V5NTD is similar to its human homologous protein, ecto-5’-nucleotidase (e5NT, a.k.a. CD73), an ecto-enzyme known for its immune regulatory activity. Through decoding V5NTD protein sequence from Naja atra venom gland cDNA, we use molecular replacement to exhibit the homodimeric isoform consists of monomers with 60 kD. This research not only provides a method to purify V5NTD from crude cobra venom, but also build the first native vertebrate ecto-5’-nucleotidase structure which elucidates the ionic-interaction in V5NTD dimerization interface and different glycosylation sites from human CD73. This molecular structural-based study can assist to clarify the biological functions of V5NTD after envenomation.
壹、 緒論 1
貳、 材料與方法 6
一、 蛇毒5端核苷酸酶cDNA定序 6
(一) 蛇毒囊解剖與組織研磨 6
(二) RNA萃取與cDNA製備 6
(三) 序列比對與引子(primer)設計 7
二、 純化蛇毒5端核苷酸酶 8
(一) 快速蛋白液相層析(FPLC) 8
(二) 反相高壓液相層析鑑定(RP-HPLC) 8
(三) 蛋白質N端定序 9
三、 快速篩選生長晶體條件 9
四、 X光繞射資料收集與數據解析(data processing) 10
(一) 數據收集(data collection) 10
(二) 數據解析(data processing) 10
(三) 以分子置換的方式解析立體結構 11
(四) 立體結構模型之建立與修正 12
參、 結果 13
一、 蛇毒5端核苷酸酶序列在物種之間有高同源性 13
二、 蛇毒5端核苷酸酶結晶型為二聚體的開放態構型 15
三、 蛇毒5端核苷酸酶金屬離子螯合位與人類CD73相同 17
四、 蛇毒5端核苷酸酶醣化修飾位與人類CD73不同 18
肆、 討論 20
一、 V5NTD一級結構討論與方法優化 20
二、 蛋白質純化方式之改良 21
三、 V5NTD與hCD73蛋白質性質與結構比較 22
四、 靜電力為形成V5NTD二聚體之主要作用力 22
五、 V5NTD與hCD73的醣化修飾 23
六、 V5NTD可能有與胞膜相連的狀態 26
七、 V5NTD可能生理功能探討 28
伍、 圖表 30
圖1、V5NTD片段氨基酸序列BLAST的結果。 30
圖2、蛇毒液內各蛋白質的純化步驟。 31
圖3、V5NTD純度分析。 32
圖4、中華眼鏡蛇V5NTD cDNA與對應之氨基酸序列。 33
圖5、中華眼鏡蛇、金黃珊瑚蛇、東部菱背響尾蛇與短尾蝮之V5NTD與小鼠、人類之CD73一級結構比較與二級結構預測。 34
圖6、中華眼鏡蛇V5NTD全長之氨基酸序列與其他近似物種的親緣關係。 35
圖7、中華眼鏡蛇V5NTD結晶型態與高解析度1.9Å之電子雲密度圖。 36
圖8、中華眼鏡蛇V5NTD單體構型與細部特徵。 37
圖9、中華眼鏡蛇V5NTD藉由CTD區域之靜電力吸引形成二聚體。 38
圖10、中華眼鏡蛇V5NTD與hCD73之金屬離子螯合位。 39
圖11、中華眼鏡蛇V5NTD與hCD73之N-glycosylation位置。 40
表1、以FPLC純化蛇毒蛋白質時使用之緩衝溶液 41
表2、中華眼鏡蛇V5NTD蛋白結晶條件與X光繞射數據資訊 42
表3、中華眼鏡蛇V5NTD與人類CD73的蛋白質性質比較 43
陸、 附錄 44
附圖一、蛇毒液內各蛋白質的組成比例。 44
附圖二、東部菱背響尾蛇與短尾蝮之蛇毒5端核苷酸酶cDNA序列比對 45
附表一、英文縮寫與中文譯名對照 46
附表二、 蛇毒液內主要酵素* 47
附表三、 引子列表 48
柒、 參考資料 48
1. You, C. W., Poyarkov, N. A., and Lin, S. M. (2015) Diversity of the snail-eating snakes Pareas (Serpentes, Pareatidae) from Taiwan. Zool Scr 44, 349-361
2. 游崇瑋 (2016) 。中國小頭蛇——馬祖新紀錄種。大自然雜誌 133,44-49。
3. Chen, C.-K., Lin, C.-C., Shih, F.-Y., Chaou, C.-H., Lin, J. C.-C., Lai, T.-I., Tseng, C.-Y., and Fang, C.-C. (2015) Population-based study of venomous snakebite in Taiwan. J Acut Med 5, 38-42
4. Kasturiratne, A., Wickremasinghe, A. R., de Silva, N., Gunawardena, N. K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D. G., and de Silva, H. J. (2008) The global burden of snakebite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med 5, e218
5. Hung, D. Z. (2004) Taiwan's venomous snakebite: epidemiological, evolution and geographic differences. Trans R Soc Trop Med Hyg 98, 96-101
6. 林意凡、林曉凌、陳慶餘 (2000)。毒蛇咬傷的認識與處置。當代醫學 27,803-808。
7. 阮祺文 (2004)。急診醫學 (頁310-317)。台北市:合記圖書出版社。
8. Chang, C. C., Wei, J. W., Chuang, S. T., and Lee, C. Y. (1972) Are the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom due to phospholipase A, neurotoxin or cardiotoxin? 台灣醫學會雜誌 71, 323-327
9. 行政院衛生署 (1981)。抗神經性蛇毒多價血清(雨傘節、飯匙倩)檢驗基準(Polyvalent Neu-rotoxic Antivenin-Bungarusmulti-cinctus and Najanajaatra)。署藥字第311010號。
10. 林增記 (2009)。急性中毒救命術-AILS (頁311-319)。台北市:金名圖書有限公司。
11. Luo, Y. T., Hsu, Y. H., Lu, C. C., Hsu, C. I., Huang, G. H. (2013) Use of Antivenin of B.multicinctus and N. atra. 藥學雜誌29, 73-76
12. Bauchot, R. (1994) Snakes: A Natural History, Sterling Publishing Co., Inc., New York City, NY, USA
13. Li, S., Wang, J., Zhang, X., Ren, Y., Wang, N., Zhao, K., Chen, X., Zhao, C., Li, X., Shao, J., Yin, J., West, M. B., Xu, N., and Liu, S. (2004) Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys. Biochem J 384, 119-127
14. Huang, H. W., Liu, B. S., Chien, K. Y., Chiang, L. C., Huang, S. Y., Sung, W. C., and Wu, W. G. (2015) Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation. J Proteomics 128, 92-104
15. Albuquerque, E. X., and Lee, C. Y. (1979) Snake venoms, Springer-Verlag, Berlin ; New York
16. Aird, S. D. (2002) Ophidian envenomation strategies and the role of purines. Toxicon 40, 335-393
17. McGivern, J. J., Wray, K. P., Margres, M. J., Couch, M. E., Mackessy, S. P., and Rokyta, D. R. (2014) RNA-seq and high-definition mass spectrometry reveal the complex and divergent venoms of two rear-fanged colubrid snakes. BMC Genomics 15, 1061
18. Moir, T. W., and Jones, P. K. (1973) Observations on the effect of changes in arterial oxygenation on adenosine induced coronary vasodilation. Adv Exp Med Biol 39, 11-26
19. Berne, R. M., and Rubio, R. (1974) Regulation of coronary blood flow. Adv Cardiol 12, 303-317
20. Kawashima, Y., Nagasawa, T., and Ninomiya, H. (2000) Contribution of ecto-5'-nucleotidase to the inhibition of platelet aggregation by human endothelial cells. Blood 96, 2157-2162
21. Salmi, M., and Jalkanen, S. (2005) Cell-surface enzymes in control of leukocyte trafficking. Nat Rev Immunol 5, 760-771
22. Chen, J. F., Eltzschig, H. K., and Fredholm, B. B. (2013) Adenosine receptors as drug targets--what are the challenges? Nat Rev Drug Discov 12, 265-286
23. Katkar, G. D., Sundaram, M. S., NaveenKumar, S. K., Swethakumar, B., Sharma, R. D., Paul, M., Vishalakshi, G. J., Devaraja, S., Girish, K. S., and Kemparaju, K. (2016) NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction. Nat Commun 7, 11361
24. Ferrari, D., McNamee, E. N., Idzko, M., Gambari, R., and Eltzschig, H. K. (2016) Purinergic Signaling During Immune Cell Trafficking. Trends Immunol 37, 399-411
25. Kaye, M. A. (1955) The effect of zinc on 5-nucleotidase of cobra venom and the interference of other nucleotides. Biochim Biophys Acta 18, 456-458
26. Lin, R. H., and Lin-Shiau, S. Y. (1982) Effects of divalent cations on 5'-nucleotidase activity of Formosan cobra venom. 台灣醫學會雜誌81, 1338-1347
27. Thompson, L. F., Eltzschig, H. K., Ibla, J. C., Van De Wiele, C. J., Resta, R., Morote-Garcia, J. C., and Colgan, S. P. (2004) Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia. J Exp Med 200, 1395-1405
28. Antonioli, L., Pacher, P., Vizi, E. S., and Hasko, G. (2013) CD39 and CD73 in immunity and inflammation. Trends Mol Med 19, 355-367
29. Knapp, K., Zebisch, M., Pippel, J., El-Tayeb, A., Muller, C. E., and Strater, N. (2012) Crystal structure of the human ecto-5'-nucleotidase (CD73): insights into the regulation of purinergic signaling. Structure 20, 2161-2173
30. Heuts, D. P., Weissenborn, M. J., Olkhov, R. V., Shaw, A. M., Gummadova, J., Levy, C., and Scrutton, N. S. (2012) Crystal structure of a soluble form of human CD73 with ecto-5'-nucleotidase activity. Chembiochem 13, 2384-2391
31. Klebe, G. (2000) Recent developments in structure-based drug design. J Mol Med (Berl) 78, 269-281
32. Yamanouye, N., Kerchove, C. M., Moura-da-Silva, A. M., Carneiro, S. M., and Markus, R. P. (2006) Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro. Nat Protoc 1, 2763-2766
33. Otwinowski, Z., Minor, W., and et al. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-326
34. David, D. E. M. a. P. R. (1999) Practical Protein Crystallography, 2 ed., Academic Press, USA
35. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221
36. UniProt, C. (2015) UniProt: a hub for protein information. Nucleic Acids Res 43, D204-212
37. Jiang, Y., Li, Y., Lee, W., Xu, X., Zhang, Y., Zhao, R., Zhang, Y., and Wang, W. (2011) Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes. BMC Genomics 12, 1
38. Tan, C. H., Tan, K. Y., Fung, S. Y., and Tan, N. H. (2015) Venom-gland transcriptome and venom proteome of the Malaysian king cobra (Ophiophagus hannah). BMC Genomics 16, 687
39. Margres, M. J., Aronow, K., Loyacano, J., and Rokyta, D. R. (2013) The venom-gland transcriptome of the eastern coral snake (Micrurus fulvius) reveals high venom complexity in the intragenomic evolution of venoms. BMC Genomics 14, 531
40. Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112, 531-552
41. Lawson, R., Slowinski, J. B., Crother, B. I., and Burbrink, F. T. (2005) Phylogeny of the Colubroidea (Serpentes): new evidence from mitochondrial and nuclear genes. Mol Phylogenet Evol 37, 581-601
42. Alam, M. S., Kuo, J. L., Ernst, P. B., Derr-Castillo, V., Pereira, M., Gaines, D., Costales, M., Bigley, E., and Williams, K. (2014) Ecto-5'-nucleotidase (CD73) regulates host inflammatory responses and exacerbates murine salmonellosis. Sci Rep 4, 4486
43. Firon, A., Dinis, M., Raynal, B., Poyart, C., Trieu-Cuot, P., and Kaminski, P. A. (2014) Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood. J Biol Chem 289, 5479-5489
44. Wang, L., Fan, J., Thompson, L. F., Zhang, Y., Shin, T., Curiel, T. J., and Zhang, B. (2011) CD73 has distinct roles in nonhematopoietic and hematopoietic cells to promote tumor growth in mice. J Clin Invest 121, 2371-2382
45. Misumi, Y., Ogata, S., Ohkubo, K., Hirose, S., and Ikehara, Y. (1990) Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form. Eur J Biochem 191, 563-569
46. Klemens, M. R., Sherman, W. R., Holmberg, N. J., Ruedi, J. M., Low, M. G., and Thompson, L. F. (1990) Characterization of soluble vs membrane-bound human placental 5'-nucleotidase. Biochem Biophys Res Commun 172, 1371-1377
47. van den Bosch, R. A., du Maine, A. P., Geuze, H. J., van der Ende, A., and Strous, G. J. (1988) Recycling of 5'-nucleotidase in a rat hepatoma cell line. EMBO J 7, 3345-3351
48. Ogawa, Y., Kanai-Azuma, M., Akimoto, Y., Kawakami, H., and Yanoshita, R. (2008) Exosome-like vesicles in Gloydius blomhoffii blomhoffii venom. Toxicon 51, 984-993
49. Dhananjaya, B. L., and D'Souza, C. J. (2010) The pharmacological role of nucleotidases in snake venoms. Cell Biochem Funct 28, 171-177
50. Teixeira, C. F., Landucci, E. C., Antunes, E., Chacur, M., and Cury, Y. (2003) Inflammatory effects of snake venom myotoxic phospholipases A2. Toxicon 42, 947-962
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔