臺灣博碩士論文加值系統

芋頭半胱胺酸蛋白酶抑制因子(tarocystatin)屬於第二群植物半胱胺酸蛋白酶抑制因子(phytocystatins)。Phytocystatins相似之處在於均具有高度保守的胺基端區域(N-terminal domain, NtD)，稱之為cystatin domain (CY domain)。不同的是第二群抑制因子比第一群在蛋白質羧基端多了胺基酸序列的延伸，但此羧基端區域(C-terminal domain, CtD)功能尚不明確。為確認NtD與CtD能否產生交互作用，藉由戊二醛交聯反應(glutaraldehyde corss-linking)及酵母菌雙雜交實驗(yeast two-hybrid)的生化方式進行分析，結果顯示NtD與CtD具有微弱的交互作用，且NtD彼此間亦會產生交互作用，使tarocystatin能形成homodimer。
由胺基酸序列比對發現NtD、CtD與已知蛋白質結構的水稻半胱胺酸蛋白酶抑制因子(oryzacystatin, OC-Ⅰ)在序列上有相似性，而蛋白質二級結構預測也發現NtD與CtD的組成相似，均由1個α-helix緊接著4個β-sheets所組成，推測CtD蛋白質的摺疊方式應與NtD類似，稱之為類CY區域(CY-like domain)，而此種二級結構組成方式亦和OC-I相同。因此以OC-Ⅰ為模板，根據交互作用實驗的結果，以同源模擬法(homology modeling)和蛋白質對接(protein-protein docking)的方式建構出兩種不同型式的tarocystatin之全長結構。
在功能分析方面，由膠體抑制活性染色實驗確認CtD具有微弱促進木瓜酶活性的能力，而全長抑制木瓜酶的效果比NtD強；又GST融合之tarocystatin會加強原始蛋白的功效，造成GST-CtD促進木瓜酶活性的能力優於CtD，而GST-全長和GST-NtD的抑制效果更佳。由本實驗的結果推論，在正常狀況下tarocystatin會以單體(monomer)及同型雙體(homodimer)的形式共存，而CtD的出現可能扮演促進tarocystatin抑制木瓜酶活性之功能。

Tarocystatin is a member of group-2 phytocystatin which could help plants against insect and pathogen attack. It shares a similar cystatin domain in the N-terminal region (NtD), and contained an extended C-terminal domain (CtD). For identifying the interaction of NtD and CtD, the glutaraldehyde cross-linking and yeast two-hybrid assays were carried out, the results showed a weak interaction between NtD and CtD, and tarocystatin could form homodimer by interaction of NtD and NtD. According to amino acids sequence alignment and secondary structure prediction, the fold of CtD should be a cystatin-like domain (CY-L domain) with similar folding to NtD which is 1 α-helix followed by 4 β-sheets. Based on domain interaction and bioinformatic analyses, we performed homology modeling and protein-protein docking to build the two models of tarocystatin using OC-I as the template. Moreover, we confirmed that CtD possess weak papain activitation property by in-gel inhibitory activity assay, full-length of tarocystatin showed higher anti-papain activity than NtD, and the GST fused tarocystatin augment their original property than none fused protein. As the results, we speculated that native tarocystatin could exist both monomeric and homodimeric form, and CtD might play a role in enhancing the inhibitory activity of tarocystatin.

目錄.....................................................I
中文摘要...............................................III
Abstract................................................IV
縮寫對照表...............................................V
第一章 前言..............................................1
1.植物之蛋白酶及蛋白酶抑制因子...........................1
2.Phytocystatin之研究與分類..............................2
3.Phytocystatin之功能....................................5
4.Phytocystatin之蛋白質結構..............................7
5.芋之簡介..............................................10
6.Tarocystatin之研究目的................................11
第二章 材料與方法.......................................12
1.材料..................................................12
2.方法..................................................12
(一)Tarocystatin胺基酸序列分析和二級結構預測............12
(二)Tarocystatin不同片段重組蛋白質之表現及純化..........13
(1)CeCPI基因表現載體之構築..............................13
(2)大腸桿菌勝任細胞之製備...............................13
(3)大腸桿菌轉形作用.....................................14
(4)重組蛋白質之大量表現及純化...........................14
(5)蛋白質之濃縮及定量分析...............................15
(6)SDS-聚丙烯醯胺膠體電泳(SDS-polyacrylamide gel electrophoresis, SDS-PAGE)..............................15
(三)Tarocystatin不同蛋白質片段之生化分析................15
(1)Glutaraldehyde (GA) cross-linking分析................15
(2)西方點墨法(western blot)分析.........................16
(3)酵母菌雙雜交(yeast two-hybrid)分析...................16
(四)Tarocystatin蛋白質結構模型之建立與分析..............17
(1)Tarocystatin蛋白質結構模型之建立.....................17
(2)Tarocystatin蛋白質結構之分析.........................17
(五)Tarocystatin不同蛋白質片段之抑制活性分析............18
第三章 結果.............................................19
1.Tarocystatin序列比對和二級結構分析....................19
2.不同片段tarocystatin之GA cross-linking分析........... 19
3.不同片段tarocystatin之yeast two-hybrid分析............21
4.Tarocystatin蛋白質結構之建立..........................22
5.Tarocystatin蛋白質結構之比對分析......................24
6.不同片段之GST-tarocystatin與tarocystatin抑制活性分析..24
第四章 討論.............................................26
1.不同片段tarocystatin的蛋白質純化結果及交互作用分析....26
2.Tarocystatin蛋白質結構分析............................27
3.GST-tarocystatin與tarocystatin功能差異之探討..........28
4.Tarocystatin CtD在功能及演化上的探討..................30
第五章 結論.............................................32
參考文獻................................................34
圖表....................................................47
附錄....................................................67

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Abe, K., Emori, Y., Kondo, H., Arai, S., and Suzuki, K. (1988). The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J. Biol. Chem. 263: 7655-7659.
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Abe, M., Abe, K., Kuroda, M., and Arai, S. (1992). Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Molecular cloning and expression studies. Eur. J. Biochem. 209: 933-937.
Abe, M., and Arai, S. (1985). Purification of a cysteine proteinase inhibitors from rice, Oryza sativa L. japonica. Agric. Biol. Chem. 49: 3349-3350.
Abe, M., Arai, S., Kato, H., and Fujimaki, M. (1980). Thiol-protease inhibitors occurring in endosperm of corn. Agric. Biol. Chem. 44: 685-686.
Abrahamson, M., Ritonja, A., Brown, M.A., Grubb, A., Machleidt, W., and Barrett, A.J. (1987). Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J. Biol. Chem. 262: 9688-9694.
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Alvarez-Fernandez, M., Barrett, A.J., Gerhartz, B., Dando, P.M., Ni, J., and Abrahamson, M. (1999). Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site. J. Biol. Chem. 274: 19195-19203.
Arai, S., Matsumoto, I., Emori, Y., and Abe, K. (2002). Plant seed cystatins and their target enzymes of endogenous and exogenous origin. J. Agric. Food Chem. 50: 6612-6617.
Arai, S., Watanabe, H., Kondo, H., Emori, Y., and Abe, K. (1991). Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members. J. Biochem. 109: 294-298.
Argall, M.E., Bradbury, J.H., and Shaw, D.C. (1994). Amino-acid sequence of a trypsin/chymotrypsin inhibitor from giant taro (Alocasia macrorrhiza). Biochem. Biophys. Acta. 1204: 189-194.
Arimatsu, Y. (1994). Latexin: a molecular marker for regional specification in the neocortex. Neurosci. Res. 20: 131-135.
Barrett, A.J. (1987). The cystatins: a new class of peptidase inhibitors. Trends Biochem. Sci. 12: 193-196.
Belenghi, B., Acconcia, F., Trovato, M., Perazzolli, M., Bocedi, A., Polticelli, F., Ascenzi, P., and Delledonne, M. (2003). AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. Eur. J. Biochem. 270: 2593-2604.
Birk, Y., and Applebaum, S.W. (1960). Effect of soybean trypsin inhibitors on the development and midgut proteolytic activity of Tribolium castaneum larvae. Enzymologia 22: 318-326.
Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., Karshikov, A., Brzin, J., Kos, J., and Turk, V. (1988). The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J. 7: 2593-2599.
Bolter, C.J. (1993). Methyl Jasmonate Induces Papain Inhibitor(s) in Tomato Leaves. Plant Physiol. 103: 1347-1353.
Botella, M.A., Xu, Y., Prabha, T.N., Zhao, Y., Narasimhan, M.L., Wilson, K.A., Nielsen, S.S., Bressan, R.A., and Hasegawa, P.M. (1996). Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate. Plant Physiol. 112: 1201-1210.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Brzin, J., Popovic, T., Drobnic-Kosorok, M., Kotnik, M., and Turk, V. (1988). Inhibitors of cysteine proteinase from potato. Biol. Chem. Hoppe-Seyler Suppl. 369: 233-238.
Brzin, J., Ritonja, A., Popvic, T., and Turk, V. (1990). Low molecular mass protein inhibitor of cystene proteinases from soybean. Biol. Chem. Hoppe-Seyler Suppl. 371: 167-170.
Cappello, F., Gatti, E., Camossetto, V., David, A., Lelouard, H., and Pierre, P. (2004). Cystatin F is secreted, but artificial modification of its C-terminus can induce its endocytic targeting Exp. Cell Res. 297: 607-618.
Chen, R., and Weng, Z. (2003). A novel shape complementarity scoring function for protein-protein docking. Proteins 51: 397-408.
Christova, P.K., Christov, N.K., and Imai, R. (2006). A cold inducible multidomain cystatin from winter wheat inhibits growth of the snow mold fungus, Microdochium nivale. Planta 223: 1207-1218.
Corre-Menguy, F., Cejudo, F.J., Mazubert, C., Vidal, J., Lelandais-Briere, C., Torres, G., Rode, A., and Hartmann, C. (2002). Characterization of the expression of a wheat cystatin gene during caryopsis development. Plant Mol. Biol. 50: 687-698.
Dieckmann, T., Mitschang, L., Hofmann, M., Kos, J., Turk, V., Auerswald, E.A., Jaenicke, R., and Oschkinat, H. (1993). The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution. J. Mol. Biol. 234: 1048-1059.
Diop, N.N., Kidrc, M., Repellin, A., Gareil, M., D''Arcy-Lameta, A., Pham Thi, A.T., and Zuily-Fodil, Y. (2004). A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves. FEBS Lett. 577: 545-550.
Dixon, D.P., Lapthorn, A., and Edwards, R. (2002). Plant glutathione transferases. Genome Biol 3: REVIEWS3004.
Fernandes, K.V.S., Campos, F.A.P., Val, R.R.D., and Filho, J.X. (1991). The expression of papain inihibitors during development of cowpea seeds. Plant Sci. 74: 179-184.
Filho, J.X. (1992). The biological roles of serine and cysteine proteinase inhibitors in plants. R. Bras. Fisiol. Veg. 4: 1-6.
Gutierrez-Campos, R., Torres-Acosta, J.A., Saucedo-Arias, L.J., and Gomez-Lim, M.A. (1999). The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nature Biotech. 17: 1223-1226.
Hall, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95-98.
Hatanaka, Y., Uratani, Y., Takiguchi-Hayashi, K., Omori, A., Sato, K., Miyamoto, M., and Arimatsu, Y. (1994). Intracortical regionality represented by specific transcription for a novel protein, latexin. Eur. J. Neurosci. 6: 973-982.
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