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系統識別號 U0026-0812200915183590
論文名稱(中文) 藉由醣質體-蛋白質體學方法分析 口腔癌細胞表面之氮基醣鍵結
論文名稱(英文) Characterization of N-linked carbohydrate chains for oral cancer by glycoproteomic analysis
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 97
學期 2
出版年 98
研究生(中文) 鄭筑文
研究生(英文) Chu-wen Cheng
電子信箱 T3696109@mail.ncku.edu.tw
學號 t3696109
學位類別 碩士
語文別 中文
論文頁數 63頁
口試委員 指導教授-張權發
口試委員-陳玉玲
口試委員-林尊湄
口試委員-靳應臺
中文關鍵字 醣結構  醣質體 
英文關鍵字 glycan structure  glycomic 
學科別分類
中文摘要 目前已知癌細胞或宿主細胞表面的醣類結構改變在癌症的病程上扮演了重要的角色,其中又以岩藻醣(fucose)以及唾液酸(sialic acid)的改變最為常見,為了研究這些複雜的醣類結構,目前已有許多醣質體學以及醣蛋白質體學的方法可以幫助分析。在我們的研究中,我們希望分析口腔癌細胞株(OC2)表面的醣結構,此外我們並選用了一株人類正常口腔角質細胞HOK來幫助比較癌細胞及正常細胞間的差異;首先我們利用目前現有的醣類抗體包括:Lex、Ley、Lea、Leb、sLex、 sLea、Tn和sTn來分析,結果顯示在OC2表面具有高量的Ley表現;此外我們也使用9種凝集素來分析(分別為AIA、ECA、LcH、Lotus、MAA、MPA、SNA、TL以及VVA),由凝集素分析的結果顯示OC2表面帶有許多Fucα1-6GlcNAc-Asn、末端Galβ1-4GlcNAc (terminal LacNAc)、末端半乳糖(terminal galactose)、T antigen (Galβ1-3GalNAc)以及GalNAc。此外由於醣類結構的改變會直接受到醣基轉移酶(glycosyltransferases)的調控,因此醣基轉移酶的活性以及表現量是影響醣類結構的關鍵,其中岩藻醣及唾液酸則是分別由fucosyltransferase (FucTs)以及sialyltransferases (SiaTs)所調控,所以我們更進一步利用real time PCR分析這些醣基轉移酶的mRNA表現量,發現在OC2中FUT8的mRNA表現量約為HOK的三倍之多,因此我們預期將會在OC2表面醣類觀察到許多α1-6 core fucosylation的結構;此外由於OC2的FUT4和FUT5的mRNA表現量皆低於HOK,因此預期在CO2表面會有較少的α 1-3鍵結的岩藻醣;最後我們取得N-linked glycans,經由全甲基化步驟處理後利用質譜儀來分析,發現OC2表面醣類確實含有許多fucose,而更進一步的鍵結分析也將在之後的MS/MS分析後完成。
英文摘要 Specific glycans expressed on tumor or host surface have now been identified as mediating key pathophysiological events during the various progression steps of tumor. Most of the glycan structures on the surface of cancer cell are altered and these modifications have been influenced and controlled by the expression and activity of glycosyltransferases, especially fucosyltransferases (FucTs) and sialyltransferases (SiaTs). Hence, the structure and function of these surface carbohydrates have attracted a lot of attention and many important glycomic and glycoproteomic techniques have also been developed. In this study, we are going to characterize the surface N-linked glycans structures of oral cancer cells (OC2). In addition, we also compared the OC2 with HOK, which is a normal human oral keratinocytes. The surface glycan was first analyzed with eight cancer associated antigens (Lex, Ley, Lea, Leb, sLex, sLea, Tn and sTn) and lectins (AIA, ECA, LcH, Lotus, MAA, MPA, SNA, TL and VVA) by flow cytometry. The result had shown that Ley shifted majorly, and glycans of oral cancer cell may include lots of Fucα1-6GlcNAc-Asn, terminal LacNAc, terminal galactose, T antigen and GalNAc. Further, we had used real time PCR to determine the amount of glycosyltransferases mRNA. The result showed that the mRNA expression level of FUT8 in OC2 is three fold higher than HOK. Therefore, we purpose there should have a lot of α1-6 core fucosylation structure on the surface of OC2. The mRNA expression level of FUT4 and FUT5 in OC2 is lower than HOK. Therefore, we purpose there should have fewer α1-3 core fucosylation structures on the surface of OC2. In the final step, the membrane proteins of OC2 were extracted and the N-linked glycans were released. Following with permethylation, the N-linked glycans were subjected to MALDI-mass spectrometry for structure analysis.
論文目次 Abstract 1
中文摘要 3
致謝 4
目錄 5
圖目錄 7
第一章 研究背景 8
1-1前言 8
1-2醣類的結構及生合成 8
1-3醣化作用與癌症 10
1-4口腔癌 11
1-5醣質蛋白質體學(glycoproteomics) 12
1-5-1醣質蛋白質體學 12
1-5-2研究醣質蛋白質體學之方法(Glycoproteomics Approach) 12
第二章 研究目標 15
第三章 研究方法與材料 17
3-1細胞培養 17
3-1-1人類口腔癌細胞株OC2 17
3-1-2人類正常口腔細胞NOK與HOK 17
3-2螢光染色 17
3-3萃取細胞膜蛋白 18
3-4蛋白質定量 18
3-5醣類萃取 18
3-6全甲基化(Permethylation) 19
3-7利用流式細胞儀做醣類抗體分析 19
3-8利用流式細胞儀做凝集素分析 20
3-9細胞RNA萃取 20
3-10反轉錄反應(Reverse transcription) 20
3-11即時定量聚和酶連鎖反應(real-time PCR) 21
第四章 結果 22
4-1 Fucosyltransferase的mRNA在臨床分離的口腔正常細胞NOK以及癌細胞株OC2中的相對表現量 22
4-2 Fut1,2和Fut4在臨床配對檢體中的相對表現量 22
4-3 Glycosyltransferases的mRNA在人類口腔角質細胞株(HOK)以及癌細胞株OC2中的相對表現量 22
4-4利用Metabolic Carbohydrate Labeling觀察到OC2和HOK上醣類影像 23
4-5 利用流式細胞儀及醣類抗體分析OC2與HOK表面醣類的差異 23
4-6 利用流式細胞儀及凝集素分析OC2與HOK表面醣類的差異 23
4-7分析OC2表面N-linked glycans之結構 24
4-8 分析OC2表面O-linked glycans之結構 24
4-9 分析HOK表面N-linked glycans之結構 24
第五章 討論 25
參考文獻 28
附圖表 33
附錄 59
附錄一 Metabolic Carbohydrate Labeling原理 59
附錄二 Lectin所辨認之醣結構 60
附錄三 縮寫表 61
參考文獻 Apweiler, R., Hermjakob, H., and Sharon, N. (1999). On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta 1473, 4-8.
Bond, M.R., and Kohler, J.J. (2007). Chemical methods for glycoprotein discovery. Curr Opin Chem Biol 11, 52-58.
Comunale, M.A., Lowman, M., Long, R.E., Krakover, J., Philip, R., Seeholzer, S., Evans, A.A., Hann, H.W., Block, T.M., and Mehta, A.S. (2006). Proteomic analysis of serum associated fucosylated glycoproteins in the development of primary hepatocellular carcinoma. J Proteome Res 5, 308-315.
Dall'Olio, F., Malagolini, N., di Stefano, G., Minni, F., Marrano, D., and Serafini-Cessi, F. (1989). Increased CMP-NeuAc:Gal beta 1,4GlcNAc-R alpha 2,6 sialyltransferase activity in human colorectal cancer tissues. Int J Cancer 44, 434-439.
Dalpathado, D.S., and Desaire, H. (2008). Glycopeptide analysis by mass spectrometry. Analyst 133, 731-738.
Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987). Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis. Science 236, 582-585.
Dube, D.H., and Bertozzi, C.R. (2005). Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov 4, 477-488.
Fuster, M.M., and Esko, J.D. (2005). The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 5, 526-542.
Gessner, P., Riedl, S., Quentmaier, A., and Kemmner, W. (1993). Enhanced activity of CMP-neuAc:Gal beta 1-4GlcNAc:alpha 2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Lett 75, 143-149.
Geyer, H., and Geyer, R. (2006). Strategies for analysis of glycoprotein glycosylation. Biochim Biophys Acta 1764, 1853-1869.
Glozman, R., Okiyoneda, T., Mulvihill, C.M., Rini, J.M., Barriere, H., and Lukacs, G.L. (2009). N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. J Cell Biol 184, 847-862.
Goelz, S.E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990). ELFT: a gene that directs the expression of an ELAM-1 ligand. Cell 63, 1349-1356.
Gu, J., and Taniguchi, N. (2008). Potential of N-glycan in cell adhesion and migration as either a positive or negative regulator. Cell Adh Migr 2, 243-245.
Hasegawa, H., Watanabe, M., Arisawa, Y., Teramoto, T., Kodaira, S., and Kitajima, M. (1993). Carbohydrate antigens and liver metastasis in colorectal cancer. Jpn J Clin Oncol 23, 336-341.
Helenius, A., and Aebi, M. (2001). Intracellular functions of N-linked glycans. Science 291, 2364-2369.
Helenius, A., and Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73, 1019-1049.
Hsu, T.L., Hanson, S.R., Kishikawa, K., Wang, S.K., Sawa, M., and Wong, C.H. (2007). Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci U S A 104, 2614-2619.
Hutchinson, W.L., Du, M.Q., Johnson, P.J., and Williams, R. (1991). Fucosyltransferases: differential plasma and tissue alterations in hepatocellular carcinoma and cirrhosis. Hepatology 13, 683-688.
Iozzo, R.V. (2005). Basement membrane proteoglycans: from cellar to ceiling. Nat Rev Mol Cell Biol 6, 646-656.
Kaneko, M., Kudo, T., Iwasaki, H., Shiina, T., Inoko, H., Kozaki, T., Saitou, N., and Narimatsu, H. (1999). Assignment of the human alpha 1,3-fucosyltransferase IX gene (FUT9) to chromosome band 6q16 by in situ hybridization. Cytogenet Cell Genet 86, 329-330.
Kang, P., Mechref, Y., and Novotny, M.V. (2008). High-throughput solid-phase permethylation of glycans prior to mass spectrometry. Rapid Commun Mass Spectrom 22, 721-734.
Kelly, R.J., Rouquier, S., Giorgi, D., Lennon, G.G., and Lowe, J.B. (1995). Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltransferase gene (FUT2). Homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype. J Biol Chem 270, 4640-4649.
Larsen, R.D., Ernst, L.K., Nair, R.P., and Lowe, J.B. (1990). Molecular cloning, sequence, and expression of a human GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase cDNA that can form the H blood group antigen. Proc Natl Acad Sci U S A 87, 6674-6678.
Lowe, J.B. (2001). Glycosylation, immunity, and autoimmunity. Cell 104, 809-812.
Lowe, J.B., Kukowska-Latallo, J.F., Nair, R.P., Larsen, R.D., Marks, R.M., Macher, B.A., Kelly, R.J., and Ernst, L.K. (1991). Molecular cloning of a human fucosyltransferase gene that determines expression of the Lewis x and VIM-2 epitopes but not ELAM-1-dependent cell adhesion. J Biol Chem 266, 17467-17477.
Mahal, L.K., Yarema, K.J., and Bertozzi, C.R. (1997). Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125-1128.
Malagolini, N., Santini, D., Chiricolo, M., and Dall'Olio, F. (2007). Biosynthesis and expression of the Sda and sialyl Lewis x antigens in normal and cancer colon. Glycobiology 17, 688-697.
Manoharan, S., Padmanabhan, M., Kolanjiappan, K., Ramachandran, C.R., and Suresh, K. (2004). Analysis of glycoconjugates in patients with oral squamous cell carcinoma. Clin Chim Acta 339, 91-96.
Matsui, T., Kojima, H., Suzuki, H., Hamajima, H., Nakazato, H., Ito, K., Nakao, A., and Sakamoto, J. (2004). Sialyl Lewisa expression as a predictor of the prognosis of colon carcinoma patients in a prospective randomized clinical trial. Jpn J Clin Oncol 34, 588-593.
McCurley, R.S., Recinos, A., 3rd, Olsen, A.S., Gingrich, J.C., Szczepaniak, D., Cameron, H.S., Krauss, R., and Weston, B.W. (1995). Physical maps of human alpha (1,3)fucosyltransferase genes FUT3-FUT6 on chromosomes 19p13.3 and 11q21. Genomics 26, 142-146.
Mitoma, J., Bao, X., Petryanik, B., Schaerli, P., Gauguet, J.M., Yu, S.Y., Kawashima, H., Saito, H., Ohtsubo, K., Marth, J.D., et al. (2007). Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment. Nat Immunol 8, 409-418.
Morelle, W., and Michalski, J.C. (2007). Analysis of protein glycosylation by mass spectrometry. Nat Protoc 2, 1585-1602.
Muinelo-Romay, L., Vazquez-Martin, C., Villar-Portela, S., Cuevas, E., Gil-Martin, E., and Fernandez-Briera, A. (2008). Expression and enzyme activity of alpha(1,6)fucosyltransferase in human colorectal cancer. Int J Cancer 123, 641-646.
Petretti, T., Kemmner, W., Schulze, B., and Schlag, P.M. (2000). Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases. Gut 46, 359-366.
Price, N.P. (2008). Permethylation linkage analysis techniques for residual carbohydrates. Appl Biochem Biotechnol 148, 271-276.
Roseman, S. (2001). Reflections on glycobiology. J Biol Chem 276, 41527-41542.
Sampathkumar, S.G., Li, A.V., Jones, M.B., Sun, Z., and Yarema, K.J. (2006). Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat Chem Biol 2, 149-152.
Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S., Hanai, N., and Nishi, T. (1994). Expression cloning of a novel alpha 1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes. J Biol Chem 269, 14730-14737.
Sasaki, K., Watanabe, E., Kawashima, K., Sekine, S., Dohi, T., Oshima, M., Hanai, N., Nishi, T., and Hasegawa, M. (1993). Expression cloning of a novel Gal beta (1-3/1-4) GlcNAc alpha 2,3-sialyltransferase using lectin resistance selection. J Biol Chem 268, 22782-22787.
Steplewska-Mazur, K., Gabriel, A., Zajecki, W., Wylezol, M., and Gluck, M. (2000). Breast cancer progression and expression of blood group-related tumor-associated antigens. Hybridoma 19, 129-133.
Takahashi, M., Tsuda, T., Ikeda, Y., Honke, K., and Taniguchi, N. (2004). Role of N-glycans in growth factor signaling. Glycoconj J 20, 207-212.
Taketa, K., Endo, Y., Sekiya, C., Tanikawa, K., Koji, T., Taga, H., Satomura, S., Matsuura, S., Kawai, T., and Hirai, H. (1993). A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepatocellular carcinoma. Cancer Res 53, 5419-5423.
Vigerust, D.J., and Shepherd, V.L. (2007). Virus glycosylation: role in virulence and immune interactions. Trends Microbiol 15, 211-218.
Wada, Y., Azadi, P., Costello, C.E., Dell, A., Dwek, R.A., Geyer, H., Geyer, R., Kakehi, K., Karlsson, N.G., Kato, K., et al. (2007). Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study. Glycobiology 17, 411-422.
Weston, B.W., Smith, P.L., Kelly, R.J., and Lowe, J.B. (1992). Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes. J Biol Chem 267, 24575-24584.
Wilson, J.R., Williams, D., and Schachter, H. (1976). The control of glycoprotein synthesis: N-acetylglucosamine linkage to a mannose residue as a signal for the attachment of L-fucose to the asparagine-linked N-acetylglucosamine residue of glycopeptide from alpha1-acid glycoprotein. Biochem Biophys Res Commun 72, 909-916.
Wong, D.Y., Chang, K.W., Chen, C.F., and Chang, R.C. (1990). Characterization of two new cell lines derived from oral cavity human squamous cell carcinomas--OC1 and OC2. J Oral Maxillofac Surg 48, 385-390.
Yanagidani, S., Uozumi, N., Ihara, Y., Miyoshi, E., Yamaguchi, N., and Taniguchi, N. (1997). Purification and cDNA cloning of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT) from human gastric cancer MKN45 cells. J Biochem 121, 626-632.
Yoshimura, M., Ihara, Y., Matsuzawa, Y., and Taniguchi, N. (1996). Aberrant glycosylation of E-cadherin enhances cell-cell binding to suppress metastasis. J Biol Chem 271, 13811-13815.
Yousefi, S., Higgins, E., Daoling, Z., Pollex-Kruger, A., Hindsgaul, O., and Dennis, J.W. (1991). Increased UDP-GlcNAc:Gal beta 1-3GaLNAc-R (GlcNAc to GaLNAc) beta-1, 6-N-acetylglucosaminyltransferase activity in metastatic murine tumor cell lines. Control of polylactosamine synthesis. J Biol Chem 266, 1772-1782.
Yu, L.G., Andrews, N., Zhao, Q., McKean, D., Williams, J.F., Connor, L.J., Gerasimenko, O.V., Hilkens, J., Hirabayashi, J., Kasai, K., et al. (2007). Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion. J Biol Chem 282, 773-781.
Zhao, Y., Itoh, S., Wang, X., Isaji, T., Miyoshi, E., Kariya, Y., Miyazaki, K., Kawasaki, N., Taniguchi, N., and Gu, J. (2006). Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions. J Biol Chem 281, 38343-38350.
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