臺灣博碩士論文加值系統

結合蛋白是一群位於細胞表面的蛋白質，能與細胞外間質有交互作用，這作用控制細胞生長、分化與遷移，牽涉到許多疾病的進程。結合蛋白alphaIIb beta3是大量表現於血小板表面的醣蛋白，能與血槳中在血栓形成及止血作用有重要角色的血纖維蛋白原結合。這樣的結合可以被含有RGD (Arg-Gly-Asp)序列的去結合蛋白所抑制。去結合蛋白是在蛇毒中發現的一群血小板抑制劑。它們是可溶於水的短peptide，長度從47至84個胺基酸並且具有一個RGD環，含有4到7對雙硫鍵。我們使用的去結合蛇毒蛋白(Rho)是從馬來蝮蛇蛇毒中純化出來，可阻斷血纖維蛋白和血小板上結合蛋白的結合來抑制血小板的凝集。Rho為68個胺基酸含有六對雙硫鍵以及在序列49到51的RGD的胺基酸序列。我們利用核磁共振(NMR)與抑制血小板功能測定發現由酵母菌所表現出的Rho重組蛋白具有正確的摺疊(folding)與功能。重組Rho的百分之五十抑制濃度(IC50)是78nM，和天然的Rho蛋白的效率相近，而若將RGD的D突變成E (Glu)，則活性下降了863倍以上。為了要解釋樣巨大的活性下降情形，我們使用了圓二色旋光和核磁共振光譜儀來決定他們在結構的折疊上有無差別。根據我們利用NMR判定的結果，我們解出Rho及其D51E突變株的三度空間結構。由結構看來，與結合蛋白結合的RGD/E motif位於一個被一組反平行(anti-parallel)beta-sheet夾出來的9個胺基長的環(loop)。而這個RGD/E環是從蛋白質的核心(core)向外伸出，並且RGD/E的序列位於這個環的頂端以RG[D/E]M四個胺基酸形成type-I turn。但是Rho和D51E突變株在三級結構上並無明顯差異，僅在帶正電的R49及帶負電D51或E51之間的電性分離程度有些微差異。我們推測這樣因為突變造成的活性差異可能是來自於，去結合蛋白和結合蛋白交互作用的機構不同所造成。另外我們也利用NMR研究Rho的動力學。

Integrins are the principal family of cell surface proteins that interact with the extracellular matrices. These interactions control the adhesion and migration of cells, as well as the transduction of signals that regulate cell growth and differentiation. Integrin alphaIIb beta3, a platelet membrane glycoprotein, binds to fibrinogen that plays an important role in thrombosis and hemostasis. The interaction between integrin alphaIIb beta3 and fibrinogen could be inhibited by disintegrins in an RGD (Arg-Gly-Asp)-dependent mechanism.
Snake venom disintegrins are a family of platelet aggregation inhibitors. They are short soluble peptides ranging from 47 to 84 amino acids with an RGD loop containing 4-7 disulfide bonds. Rhodostomin (Rho) is a snake venom protein isolated from Calloselasma rhodostoma. Rho is disintegrin which inhibits platelet aggregation by blocking the binding of fibrinogen to the integrin alphaIIb beta3 of platelets. Rho consists of 68 amino acids including six disulfide bonds and an RGD sequence at position 49-51. The recombinant Rho produced in Pichia pastoris inhibits the platelet aggregation with a KI of 70nM that is potent as native Rho. However, its D51E mutant inhibits the platelet aggregation with a KI of 50�嵱. In order to understand what causes an 863-fold decrease in activity, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structural differences in the tertiary folds of Rho and its D51E mutant. By NMR studies, we have determined the 3D structures of Rho and its D51E mutant. The binding site for integrin, lies in a nine-residue loop joining two strands of beta-sheet. The RGD/E loop extends outward from the protein core with the RGD/E sequence at its apex in a four-residue (RGD/EM) type I turn. The only difference in their structures is the charge separation between positive (R49) and negative residues (D/E51). We also use NMR to study the dynamics of Rho.

中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 VIII
表目錄 X
縮寫檢索表 XI
儀器 XII
第一章 緒論
1-1 結合蛋白 (Integrin) 之介紹 1
1-2 去結合蛋白(Disintegrin)之介紹 3
1-3 馬來蝮蛇蛇毒蛋白 ( Rhodostomin ) 之簡介 5
1-4 核磁共振決定蛋白質之三維結構之介紹 5
1-5 研究動機及內容簡介 8
第二章 材料與方法
2-1 馬來蝮蛇蛇毒蛋白與其突變株蛋白之表現與純化 10
2-1-1 以Pichia pastoris酵母表現系統表現馬來蝮蛇蛇毒蛋白 10
2-1-2 以鎳離子螯合色層分析法純化 13
2-1-3 逆相高效能液相層析法（reverse-phase HPLC） 15
2-1-4 以SDS-PAGE 分析 16
2-2 馬來蝮蛇蛇毒蛋白與其突變株蛋白三級結構之研究 19
2-2-1 利用NMR方法決定蛋白質的三級結構 19
2-2-2 樣品製備 20
2-2-3 NMR光譜的測定 21
2-2-4 質子判定（Proton assignment） 21
2-2-5 循序判定（Sequential assignment） 22
2-2-6 分子結構的限制條件 23
2-2-7 三級結構的計算 25
2-2-8 三級結構的顯示與排列(alignment) 29
2-2-9 利用核磁共振頻譜儀測定分子的動力學 30
第三章 實驗結果
3-1 馬來蝮蛇蛇毒蛋白之NMR圖譜的分析 31
3-2 馬來蝮蛇蛇毒蛋白之二級結構分析 32
3-3 馬來蝮蛇蛇毒蛋白的三級結構 32
3-4 馬來蝮蛇蛇毒蛋白突變株蛋白之結構研究 35
3-5 馬來蝮蛇蛇毒蛋白與其突變株動力學之研究 35
3-6 一些已經存放於INTERNET上資料庫的結果 35
第四章 討論
4-1 馬來蝮蛇蛇毒蛋白與其他蛇毒蛋白結構的比較 37
4-2 pH值對Rhodostomin在結構上所造成的影響 37
4-3 Rhodostomin與其突變株之間的差異 38
第五章 結論 40
參考文獻 41
圖 45
表 72
自述 79

Alder M., Lazarus R. A., Dennis M. S., Wagner G. Solution structure of kistrin, a potent platelet aggregation inhibitor and GP IIb-IIIa antagonist. (1991) Science 253: 445-448.
Arnaout, M.A.: Leukocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol Rev. 114: 145-180, 1990
Chiang, H.S., Yang, R.S., Huang T.F. The Arg-Gly-Asp-containing peptide, rhodostomin, inhibits in vitro cell adhesion to extracellular matrices and platelet aggregation caused by saos-2 human osteosarcoma cells.(1995) Br J Cancer 71(2):265-70.
Chang, H.H., Hu, S.T., Huang, T.F., Chen, S.H., Lee, Y.H., Lo, S.J.: Rhodostomin, an RGD-containing peptide expressed from a synthetic gene in Escherichia coli, facilitates the attachment of human hepatoma cells. Biochemical & Biophysical Research Communications 190: 242-9, 1993.
Dennis, M.S., Carter, P., Lazarus, R.A.: Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. Proteins 5: 312-321, 1993.
Williams, M. J., Hughes, P. E., O’Toole, T. E., Ginsberg, M. H. (1994) The inner world of cell adhesion: integrin cytoplasmic domains. Trends in Cell Biology 4:109-112.
Gould R. J., Polokoff M. A., Friedman P. A., Huang T. F., Holt J. C., Cook J. J., Neiwiarowski S. Disintegrins : A family of integrin inhibitory proteins from viper venoms. (1990) Proc Soc Exp Biol 195: 168-171.
Guo, R.T., Chou, L.J., Chen, Y.C., Chen, C.Y., Pari, K., Jen, CY.J., Lo, SC.J., Huang, S.L., Lee, C.Y., Chang, T.W., Chuang, W.J.: Expression in Pichia pastoris and Characterization by Circular Dichroism and NMR of Rhodostomin. Proteins. 43: 499-508, 2001
Huang, T. F., Sheu, J. R., Teng, C. M., Chen, S. W., Liu, C. S. (1991) Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex. Journal of Biochemistry 109:328-334.
Hantgan, R.R., Rocco, M., Nagaswami, C., Weisel, J.W.: Binding of a fibrinogen mimetic stabilizes integrin �圂Ib��3's open conformation. Protein Sci 10: 1614-1626, 2001.
Humphries, M.J.: Integrin structure. Biochem Soc Trans 28: 311-339, 2000.
Hynes, R.O.: Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 11–25, 1992
Juliano D., Wang Y., Marcinkiewicz C., Rosenthal L. A., Stewart G. J., Niewiaroswki S. Disintegrin interaction with alpha V beta 3 integrin on human umbilical vein endothelial cells: expression of ligand-induced binding site on beta 3 subunit. (1996) Exp Cell Res 225(1):132-42.
Knudsen K. A., Tuszynski G. P., Huang T. F., Niewiarowski S. Trigramin, an RGD-containing pepetide from snake venom, inhibits cell-substratum adhesion of human melanoma cells. (1988) Exp Cell Res 179(1):42-49.
Mahesh J. K., John S., Xinjie L. U., Janice A. W., Hylary T., Ian P. T., Eva I. H. 1H-NMR studies and secondary structure of the RGD-containing snake toxin, albolabrin. (1993) European Journal of Biochemistry 218:853-860.
Marcinkiewicz C., Calvete J. J., Niewiarowski S. Isolation and characterization of two novel dimeric disintegrins: EC-3 and EMF-10 inhibiting interaction of lymphoid cells with VCAM-1. (1999) Blood 90: 59b.
Matter M. L., Zhang Z., Nordstedt C., Rouslahti E. The alpha5beta1 integrin meditates elimination of amyloid-beta peptide and protects against apoptosis. (1998) Journal of Cell Biology 141(4): 1019-1030.
Morris V. L., Schmidt E. E., Koop S., MacDonald I., Grattan M., Khokha R., McLane M. A., Niewiarowski S., Chambers A. F., Groom A. C. Effects of the disintegrin eristostatin on individual steps of hematogenous metastatis. (1995) Exp Cell Res 219(2): 571-578.
Musial J., Niewiarowski S., Rucinski B., Stewart G. J., Cook J. J., Williams J. A., Edmunds L. H. Jr. Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins. RGD-containing peptides from viper venoms. (1990) Circulation (1): 261-273.
Peng, J.W., & Wagner, G.: Methods in Enzymol. 239:563-596, 1994.
Wuthrich, K.: NMR of Proteins and Nucleic Acid. p26-27., 1986.
Peter, L. B., Sherron, B., Stuart, B., Daniel, J. B., Kathryn, S. C., Tracy, D., Charles, E., Thomas, R., Gadek, R. G., Michael, T., Lipari, C. D. M., Mary, A. N., Robert, M. P., Allan, P., Clifford, Q., Mark, S., Martin, S., Jeffrey, Y. K. T., John, P. B. (1992) Cyclic RGD peptide analogues as antiplatelet antithrombotics. J. Med. Chem. 35:2040-2048.
Pfaff M., McLane M. A., Beviglia L., Niewiarowski S., Timpl R. Comparison of Disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition. (1994) Cell Adhes Commun 2(6): 491-501.
Rouslahti E. RGD and other recognition sequences for integrins. (1996) Annu Rev Cell Dev Biol 12:697-715.
Sastry, S. K., Horwitz, A. F. (1993) Integrin cytoplasmic domains: modulators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Current Opinion in Cell Biology 5:819-831.
Scarborough, R.M., Rose, J.W., Hsu, M.A., Philips, D.R., Fried, V.A., Campbell, A.M., Nannizzi, L., Charo, I.F.: Barbourin. A GPIIb-IIIa-specific integrin anatagonist from the venom of Sistrurus m, barbouri. J Biol Chem 266: 9359-9362, 1991.
Smyth, S. S., Joneckis, C. C., Parise, L. V. (1993) Regulation of vascular integrins. Blood 81:2827-2843.
Tcheng J. E., Harrington R. A., Kottke M. K., Kleinman N., Ellis S. G., Kereiakes D. J., Mick M. J., Navetta F. I., Smith J. E., Worley S. J., Miller J. A., Joseph D. M., Sigmon K. N., Kitt M. M., Califf R. M., Topol E. J. Multicenter , randomized, double-blind, placebo-controlled trial of the platelet integrin glycoprotein IIb/IIIa blocker integrin in elective coronary intervention. (1995) Circulation 91: 2151-2157.
Trikha M., De Clerck Y. A., Markland F. S. Contortrostatin, a snake venom disintegrin, inhibits beta 1 integrin-meditated human metastatic melanoma cell adhesion and blocks experimental metastasis. (1994) Cancer Research 54(18):4993-4998.
Vesteweber D., Blanks J. E. Mechanism that regulate the function of the selectins and their ligands.
Vladimir Daudek, R., Andrew Atkinson, and John T. P. Three-Dimensional Structure of Echistatin, the Smallest Active RGD Protein. (1991) Biochemistry 30: 7369-7372.
Xiong, J.P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D.L., Joachimiak, A., Goodman, S.L., Arnaout, M.A.: Crystal Structure of the Extracellular Segment of Integrin �埐��3. Science. 2001 Sep 6 [epub ahead of print]
Williams M. J., Hughes P. E., O’Toole T. E., Ginsberg M. H. The inner world of cell adhesion: integrin cytoplasmic domains. (1994) Trends in Cell Biology 4: 109-112.
陳金榜， 蛋白質在水溶液中之結構-多維異核核磁共振之應用。 科儀新知， 第十五卷， 第六期， 40-46， 1994。
黃忠智及余靖， 利用核磁共振光譜決定台灣眼鏡蛇蛇毒蛋白分子的水溶液中三度空間結構。 科儀新知， 第十五卷， 第六期， 21-39， 1994。
郭瑞庭， 馬來蝮蛇蛇毒基因的選殖及蛇毒蛋白之功能及三度空間結構之研究。國立成功大學生物化學研究所碩士論文， 1998。
周立哲， 馬來蝮蛇蛇毒蛋白及其D51E突變蛋白結構的核磁共振研究。國立成功大學生物化學研究所碩士論文， 2000。