半乳糖凝集素–3是一個由250個胺基酸組成的蛋白質，包含前端一段長112個胺基酸的無結構胺基酸多肽鏈以及後端138個胺基酸的有結構碳水化合物辨認位。半乳糖凝集素–3會廣泛的分布在哺乳動物的體內；不論是循環系統中或者是細胞內、外，都可以發現半乳糖凝集素–3的存在。而半乳糖凝集素–3的生物功能也同樣廣泛，已知半乳糖凝集素–3能夠調控細胞轉化、調控細胞凋亡以及調控細胞的生命週期。而前人的研究指出，半乳糖凝集素–3這些功能的調控並不僅僅是受控於有結構的碳水化合物辨認位。研究指出位於無結構的胺基酸多肽鏈位置的突變會影響到半乳糖凝集素–3與Ras蛋白的結合，而導致細胞轉化的持續發生。而同樣在無結構的胺基酸序列的部分胺基酸的缺失會影響半乳糖凝集素–3進出細胞核的能力。還有研究指出位於無結構的胺基酸序列有突變發生時，會影響到半乳糖凝集素–3辨認乳糖的能力。這代表半乳糖凝集素–3的無結構的胺基酸序列用某種方式影響著整個半乳糖凝集素–3的功能，但關於無結構的胺基酸序列如何影響整個蛋白質的相關研究卻很少。於是我們在完整的半乳糖凝集素–3蛋白質上的無結構胺基酸序列上選定三個點（10、75、100）。因為A10靠近S6以及S12這兩個與功能有密切關係的磷酸化位點。而A100則是靠近Y107與碳水化合物辨認位辨認能力有關連。A75則是因為靠近N端無結構的中段，希望可以藉由這個點來探討N端無結構蛋白的中段結構變化。藉由核磁共振的順磁弛豫增強實驗來研究這三個點與半乳糖凝集素–3的碳水化合物辨認位是否存在交互作用。我們的研究結果顯示半乳糖凝集素–3的無結構胺基酸序列部分存在有與碳水化合物辨認位交互作用的傾向；以及無結構的胺基酸序列本身可能也存在互相靠近的傾向。

Galectin-3 is the protein consisted of 112 disordered amino acids sequence of N-termini and 138 structural amino acids sequence of C-terminal carbohydrate recognition domain (CRD). Galectin-3 is widespread on mammalians body; including nuclear, plasma and circulatory system. Galectin-3 also has the wide biofunctions. We know that galectin-3 may involve the cell transformation, regulation of cell apoptosis and regulation of cell cycle. Some papers reported that disordered N-termini of galectin-3 may affect biofunctions of galectin-3. The papers showed that mutations on galectin-3 disordered N-termini avoid binding of galectin-3 and K-Ras protein, it will lead the cell continuously transform. And deletions on disordered N-termini of galectin-3 make the galectin-3 loss ability of transportation from nuclear to plasma. On the other research said that the lactose binging affinity of galectin-3 decreased as mutations on N-termini of galectin-3. It shows that N-termini of galectin-3 affects the protein functions by some interactions, but there are little research about N- and C- interaction. We want to know how the interaction between N- and C-terminus. We make three cysteines to alanines mutantion on disordered N-terminal of galectin-3, galetin-3-A10C, -A75C and -A100C. We made the NMR paramagnetic relaxation enhancement on three mutants. On this research, we found that disordered N-terminal of galectin-3 may interact with CRD and disordered N-terminal of galectin-3 may have the special attention of folding.

致謝 i
英文摘要 ii
中文摘要 iii
目錄 iv
圖目錄 v
表目錄 vi
Chapter 1. 背景介紹 1
前言 1
1.1 半乳糖凝集素的介紹 4
1.2 半乳糖凝集素的生物功能與疾病 9
1.3 半乳糖凝集素–3的結構與結構對功能的影響 17
目的 22
Chapter 2. 材料與方法 23
2.1 試劑 23
2.2 儀器 29
2.3 方法與步驟 30
Chapter 3. 實驗原理與結果 42
3.1建立半乳糖凝集素–3與其突變株的質體 42
3.2 測試並建立培養突變半乳糖凝集素–3之表現標準流程 55
3.3 建立檢驗MTSL標定標準流程 67
3.4 順磁弛豫增強（paramagnetic relaxation enhancement，PRE）實驗 72
Chapter 4. 討論與結論 81
附錄 87
參考文獻 88

圖 1. 半乳糖凝集素在生物體中的重要功能示意圖 8
圖 2. 半乳糖凝集素家族參與癌細胞的轉化（transformation） 12
圖 3. 半乳糖凝集素家族對於癌細胞中的細胞凋亡的調控 13
圖 4. 半乳糖凝集素家族對於癌細胞的細胞週期的調控 16
圖 5. 半乳糖凝集素–3的結構 20
圖 6. 蛋白質SDS膠體電泳之預染色蛋白標定（marker）參考 28
圖 7. 蛋白質多肽鏈與MTSL反應以及標定 43
圖 8. 半乳糖凝集素–3–C173A以及A10C、A75C、A100C的突變株的胺基酸序列示意圖 46
圖 9. 融合蛋白SUMO-gal3-A75C-C173A之質體建構以及SUMO專一性蛋白酶ulp1c作用示意圖 49
圖 10. 以基因重組將Gal3–A75C–C173A接在pET32a–SUMO系統中 53
圖 11. 分別以Rosetta、BL–21勝任細胞表現半乳糖凝集素–3–C173A的蛋白質SDS電泳膠圖 57
圖 12. 以lactosyl – Sepharose 4B®管柱純化半乳糖凝集素–3–C173A 59
圖 13. 比較不同溫條件下之半乳糖凝集素–3–C173A之表達量 61
圖 14. 半乳糖凝集素–3–A75C–C173A在pET25b系統以及pET32a–SUMO系統下的表現 62
圖 15. 分別表現半乳糖凝集素–3–A10C∕A75C–C173A 63
圖 16. 純化SUMO–Gal3–A75C–C173A 65
圖 17. 以DTNB來辨識R–SH的原理 68
圖 18. 以DTNB檢測半乳糖凝集素–3–C173A–A10C被MTSL標定 71
圖 19. 常用於順磁弛豫增強實驗的化學藥物 76
圖 20. 半乳糖凝集素–3的順磁弛豫增強實驗結果 79
圖 21. 檢證A10C、A100C的NMR實驗樣品是否發生降解 80
圖 22. 預測的半乳糖凝集素–3之N端與C端交互作用的模型 86

表 1. 半乳糖凝集素家族的分類 6
表 2. 化學藥品 23
表 3. 同位素標記藥品 27
表 4. 試劑套組及標準樣品 27
表 5. 使用KAPA PCR kit的反應混和液配置 34
表 6. 使用DpnI酵素的反應混和液配置 34
表 7. 大腸桿菌（Escherichia coli，E. coli）之基因使用頻率表（gene codon usage） 43
表 8. 量化100毫升 LB培養液培養之半乳糖凝集素–3–C173A 61

Ahmad, N., H. J. Gabius, S. Andre, H. Kaltner, S. Sabesan, R. Roy, B. Liu, F. Macaluso and C. F. Brewer (2004). "Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes." J Biol Chem 279(12): 10841-10847.
Barboni, E. A., S. Bawumia, K. Henrick and R. C. Hughes (2000). "Molecular modeling and mutagenesis studies of the N-terminal domains of galectin-3: evidence for participation with the C-terminal carbohydrate recognition domain in oligosaccharide binding." Glycobiology 10(11): 1201-1208.
Barondes, S. H., V. Castronovo, D. N. Cooper, R. D. Cummings, K. Drickamer, T. Feizi, M. A. Gitt, J. Hirabayashi, C. Hughes, K. Kasai and et al. (1994). "Galectins: a family of animal beta-galactoside-binding lectins." Cell 76(4): 597-598.
Barondes, S. H., D. N. Cooper, M. A. Gitt and H. Leffler (1994). "Galectins. Structure and function of a large family of animal lectins." J Biol Chem 269(33): 20807-20810.
Bernerd, F., A. Sarasin and T. Magnaldo (1999). "Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes." Proc Natl Acad Sci U S A 96(20): 11329-11334.
Bordo, D., & Argos, P. (1991). "Suggestions for "safe" residue substitutions in site-directed mutagenesis." J Mol Biol. 217(4): 721-729.
Brewer, C. F., M. C. Miceli and L. G. Baum (2002). "Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions." Curr Opin Struct Biol 12(5): 616-623.
Choi, J. H., K. H. Chun, A. Raz and R. Lotan (2004). "Inhibition of N-(4-hydroxyphenyl)retinamide-induced apoptosis in breast cancer cells by galectin-3." Cancer Biol Ther 3(5): 447-452.
Collier, H. B. (1973). "Letter: A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate." Anal Biochem 56(1): 310-311.
Cooper, D. N. and S. H. Barondes (1990). "Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism." J Cell Biol 110(5): 1681-1691.
de Boer, R. A., A. A. Voors, P. Muntendam, W. H. van Gilst and D. J. van Veldhuisen (2009). "Galectin-3: a novel mediator of heart failure development and progression." Eur J Heart Fail 11(9): 811-817.
Dumic, J., S. Dabelic and M. Flogel (2006). "Galectin-3: an open-ended story." Biochim Biophys Acta 1760(4): 616-635.
Elad-Sfadia, G., R. Haklai, E. Balan and Y. Kloog (2004). "Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity." J Biol Chem 279(33): 34922-34930.
Elad-Sfadia, G., R. Haklai, E. Ballan, H. J. Gabius and Y. Kloog (2002). "Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase." J Biol Chem 277(40): 37169-37175.
Ellman, G. L. (1959). "Tissue sulfhydryl groups." Arch Biochem Biophys 82(1): 70-77.
Gong, H. C., Y. Honjo, P. Nangia-Makker, V. Hogan, N. Mazurak, R. S. Bresalier and A. Raz (1999). "The NH2 terminus of galectin-3 governs cellular compartmentalization and functions in cancer cells." Cancer Res 59(24): 6239-6245.
Henderson, N. C., A. C. Mackinnon, S. L. Farnworth, T. Kipari, C. Haslett, J. P. Iredale, F. T. Liu, J. Hughes and T. Sethi (2008). "Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis." Am J Pathol 172(2): 288-298.
Henderson, N. C., A. C. Mackinnon, S. L. Farnworth, F. Poirier, F. P. Russo, J. P. Iredale, C. Haslett, K. J. Simpson and T. Sethi (2006). "Galectin-3 regulates myofibroblast activation and hepatic fibrosis." Proc Natl Acad Sci U S A 103(13): 5060-5065.
Henderson, N. C. and T. Sethi (2009). "The regulation of inflammation by galectin-3." Immunol Rev 230(1): 160-171.
Hirabayashi, J., T. Hashidate, Y. Arata, N. Nishi, T. Nakamura, M. Hirashima, T. Urashima, T. Oka, M. Futai, W. E. Muller, F. Yagi and K. Kasai (2002). "Oligosaccharide specificity of galectins: a search by frontal affinity chromatography." Biochim Biophys Acta 1572(2-3): 232-254.
Honjo, Y., P. Nangia-Makker, H. Inohara and A. Raz (2001). "Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells." Clin Cancer Res 7(3): 661-668.
Hotta, K., T. Funahashi, Y. Matsukawa, M. Takahashi, H. Nishizawa, K. Kishida, M. Matsuda, H. Kuriyama, S. Kihara, T. Nakamura, Y. Tochino, N. L. Bodkin, B. C. Hansen and Y. Matsuzawa (2001). "Galectin-12, an Adipose-expressed Galectin-like Molecule Possessing Apoptosis-inducing Activity." J Biol Chem 276(36): 34089-34097.
Houzelstein, D., I. R. Goncalves, A. J. Fadden, S. S. Sidhu, D. N. Cooper, K. Drickamer, H. Leffler and F. Poirier (2004). "Phylogenetic analysis of the vertebrate galectin family." Mol Biol Evol 21(7): 1177-1187.
Hoyer, K. K., M. Pang, D. Gui, I. P. Shintaku, I. Kuwabara, F. T. Liu, J. W. Said, L. G. Baum and M. A. Teitell (2004). "An anti-apoptotic role for galectin-3 in diffuse large B-cell lymphomas." Am J Pathol 164(3): 893-902.
Hubbell, W. L., & Altenbach, C.A. (1994). "Investigation of structure and dynamics in membrane proteins using site-directed spin labeling." Curr Opin Struct Biol 4: 566-573
Hubbell, W. L., H. S. McHaourab, C. Altenbach and M. A. Lietzow (1996). "Watching proteins move using site-directed spin labeling." Structure 4(7): 779-783.
Hughes, R. C. (1999). "Secretion of the galectin family of mammalian carbohydrate-binding proteins." Biochim Biophys Acta 1473(1): 172-185.
Ippel, H., M. C. Miller, M. A. Berbis, D. Suylen, S. Andre, T. M. Hackeng, F. J. Canada, C. Weber, H. J. Gabius, J. Jimenez-Barbero and K. H. Mayo (2015). "(1)H, (13)C, and (15)N backbone and side-chain chemical shift assignments for the 36 proline-containing, full length 29 kDa human chimera-type galectin-3." Biomol NMR Assign 9(1): 59-63.
Ippel, H., M. C. Miller, S. Vertesy, Y. Zheng, F. J. Canada, D. Suylen, K. Umemoto, C. Romano, T. Hackeng, G. Tai, H. Leffler, J. Kopitz, S. Andre, D. Kubler, J. Jimenez-Barbero, S. Oscarson, H. J. Gabius and K. H. Mayo (2016). "Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR." Glycobiology.
Kim, H. R., H. M. Lin, H. Biliran and A. Raz (1999). "Cell cycle arrest and inhibition of anoikis by galectin-3 in human breast epithelial cells." Cancer Res 59(16): 4148-4154.
Kopitz, J., S. Andre, C. von Reitzenstein, K. Versluis, H. Kaltner, R. J. Pieters, K. Wasano, I. Kuwabara, F. T. Liu, M. Cantz, A. J. Heck and H. J. Gabius (2003). "Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells." Oncogene 22(40): 6277-6288.
Kopitz, J., C. von Reitzenstein, S. Andre, H. Kaltner, J. Uhl, V. Ehemann, M. Cantz and H. J. Gabius (2001). "Negative regulation of neuroblastoma cell growth by carbohydrate-dependent surface binding of galectin-1 and functional divergence from galectin-3." J Biol Chem 276(38): 35917-35923.
Kuwabara, I., Y. Kuwabara, R. Y. Yang, M. Schuler, D. R. Green, B. L. Zuraw, D. K. Hsu and F. T. Liu (2002). "Galectin-7 (PIG1) exhibits pro-apoptotic function through JNK activation and mitochondrial cytochrome c release." J Biol Chem 277(5): 3487-3497.
Lee, C. D., H. C. Sun, S. M. Hu, C. F. Chiu, A. Homhuan, S. M. Liang, C. H. Leng and T. F. Wang (2008). "An improved SUMO fusion protein system for effective production of native proteins." Protein Sci 17(7): 1241-1248.
Liu, F. T., R. J. Patterson and J. L. Wang (2002). "Intracellular functions of galectins." Biochim Biophys Acta 1572(2-3): 263-273.
Liu, F. T. and G. A. Rabinovich (2005). "Galectins as modulators of tumour progression." Nat Rev Cancer 5(1): 29-41.
Liu, F. T. and G. A. Rabinovich (2010). "Galectins: regulators of acute and chronic inflammation." Ann N Y Acad Sci 1183: 158-182.
Liu, Y. H., M. D'Ambrosio, T. D. Liao, H. Peng, N. E. Rhaleb, U. Sharma, S. Andre, H. J. Gabius and O. A. Carretero (2009). "N-acetyl-seryl-aspartyl-lysyl-proline prevents cardiac remodeling and dysfunction induced by galectin-3, a mammalian adhesion/growth-regulatory lectin." Am J Physiol Heart Circ Physiol 296(2): H404-412.
Lok, D. J., P. Van Der Meer, P. W. de la Porte, E. Lipsic, J. Van Wijngaarden, H. L. Hillege and D. J. van Veldhuisen (2010). "Prognostic value of galectin-3, a novel marker of fibrosis, in patients with chronic heart failure: data from the DEAL-HF study." Clin Res Cardiol 99(5): 323-328.
Mackinnon, A. C., M. A. Gibbons, S. L. Farnworth, H. Leffler, U. J. Nilsson, T. Delaine, A. J. Simpson, S. J. Forbes, N. Hirani, J. Gauldie and T. Sethi (2012). "Regulation of transforming growth factor-beta1-driven lung fibrosis by galectin-3." Am J Respir Crit Care Med 185(5): 537-546.
Maloy, S. R., Stewart, V. J., & Taylor, R. K. (1996). "Genetic analysis of pathogenic bacteria: A laboratory manual." Plainview, N.Y: Cold Spring Harbor Laboratory Press.
Matarrese, P., N. Tinari, M. L. Semeraro, C. Natoli, S. Iacobelli and W. Malorni (2000). "Galectin-3 overexpression protects from cell damage and death by influencing mitochondrial homeostasis." FEBS Lett 473(3): 311-315.
Otting, G. (2010). "Protein NMR using paramagnetic ions." Annu Rev Biophys 39: 387-405.
Paz, A., R. Haklai, G. Elad-Sfadia, E. Ballan and Y. Kloog (2001). "Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation." Oncogene 20(51): 7486-7493.
Riddles, P. W., R. L. Blakeley and B. Zerner (1983). "Reassessment of Ellman's reagent." Methods Enzymol 91: 49-60.
Rini, J. M. and Y. D. Lobsanov (1999). "New animal lectin structures." Curr Opin Struct Biol 9(5): 578-584.
Rosano, G. L. and E. A. Ceccarelli (2014). "Recombinant protein expression in Escherichia coli: advances and challenges." Front Microbiol 5: 172.
Schwarz, F. and M. Aebi (2011). "Mechanisms and principles of N-linked protein glycosylation." Curr Opin Struct Biol 21(5): 576-582.
Shalom-Feuerstein, R., T. Cooks, A. Raz and Y. Kloog (2005). "Galectin-3 regulates a molecular switch from N-Ras to K-Ras usage in human breast carcinoma cells." Cancer Res 65(16): 7292-7300.
Sharma, U. C., S. Pokharel, T. J. van Brakel, J. H. van Berlo, J. P. Cleutjens, B. Schroen, S. Andre, H. J. Crijns, H. J. Gabius, J. Maessen and Y. M. Pinto (2004). "Galectin-3 marks activated macrophages in failure-prone hypertrophied hearts and contributes to cardiac dysfunction." Circulation 110(19): 3121-3128.
Shimura, T., Y. Takenaka, S. Tsutsumi, V. Hogan, A. Kikuchi and A. Raz (2004). "Galectin-3, a novel binding partner of beta-catenin." Cancer Res 64(18): 6363-6367.
Takenaka, Y., T. Fukumori, T. Yoshii, N. Oka, H. Inohara, H. R. C. Kim, R. S. Bresalier and A. Raz (2004). "Nuclear Export of Phosphorylated Galectin-3 Regulates Its Antiapoptotic Activity in Response to Chemotherapeutic Drugs." Molecular and Cellular Biology 24(10): 4395-4406.
Takenaka, Y., H. Inohara, T. Yoshii, K. Oshima, S. Nakahara, S. Akahani, Y. Honjo, Y. Yamamoto, A. Raz and T. Kubo (2003). "Malignant transformation of thyroid follicular cells by galectin-3." Cancer Lett 195(1): 111-119.
van den Brule, F., S. Califice and V. Castronovo (2004). "Expression of galectins in cancer: a critical review." Glycoconj J 19(7-9): 537-542.
Van den Steen, P., P. M. Rudd, R. A. Dwek and G. Opdenakker (1998). "Concepts and principles of O-linked glycosylation." Crit Rev Biochem Mol Biol 33(3): 151-208.
van Kimmenade, R. R., J. L. Januzzi, Jr., P. T. Ellinor, U. C. Sharma, J. A. Bakker, A. F. Low, A. Martinez, H. J. Crijns, C. A. MacRae, P. P. Menheere and Y. M. Pinto (2006). "Utility of amino-terminal pro-brain natriuretic peptide, galectin-3, and apelin for the evaluation of patients with acute heart failure." J Am Coll Cardiol 48(6): 1217-1224.
Wells, V., D. Davies and L. Mallucci (1999). "Cell cycle arrest and induction of apoptosis by beta galactoside binding protein (beta GBP) in human mammary cancer cells. A potential new approach to cancer control." Eur J Cancer 35(6): 978-983.
Yamaoka, K., K. Mishima, Y. Nagashima, A. Asai, Y. Sanai and T. Kirino (2000). "Expression of galectin-1 mRNA correlates with the malignant potential of human gliomas and expression of antisense galectin-1 inhibits the growth of 9 glioma cells." J Neurosci Res 59(6): 722-730.
Yan, Y. P., B. T. Lang, R. Vemuganti and R. J. Dempsey (2009). "Galectin-3 mediates post-ischemic tissue remodeling." Brain Res 1288: 116-124.
Yang, R. Y., D. K. Hsu and F. T. Liu (1996). "Expression of galectin-3 modulates T-cell growth and apoptosis." Proc Natl Acad Sci U S A 93(13): 6737-6742.
Yang, R. Y. and F. T. Liu (2003). "Galectins in cell growth and apoptosis." Cell Mol Life Sci 60(2): 267-276.
Yoshii, T., H. Inohara, Y. Takenaka, Y. Honjo, S. Akahani, T. Nomura, A. Raz and T. Kubo (2001). "Galectin-3 maintains the transformed phenotype of thyroid papillary carcinoma cells." Int J Oncol 18(4): 787-792.
Yu, F., R. L. Finley, Jr., A. Raz and H. R. Kim (2002). "Galectin-3 translocates to the perinuclear membranes and inhibits cytochrome c release from the mitochondria. A role for synexin in galectin-3 translocation." J Biol Chem 277(18): 15819-15827