利用抗体亲和层析纯化不同转移潜能人肝癌细胞系中的高尔基体蛋白73(GP73),比较不同转移潜能人肝癌细胞系中GP73 糖基化修饰水平,为GP73 成为肝癌标志物提供依据。纯化的GP73 通过质谱鉴定后,分别进行蛋白印迹(western blot)和凝集素印迹(lectin blot)分析,比较其在不同转移潜能肝癌细胞系的糖基化修饰水平。实验发现,GP73 含有伴刀豆凝集素(ConA)、橙黄网胞盘菌凝集素(AAL)、小扁豆凝集素(LCA)和红腰果E 型凝集素(PHA-E)识别的糖型,且单位GP73 的LCA、PHA-E 识别的岩藻糖和平分型糖型含量在肝癌细胞Huh7、MHCC97L、MHCC97H、HCCLM3 中有升高趋势。研究表明,单位GP73 岩藻糖和平分型聚糖结构随肝癌细胞的转移潜能升高而增多。
In this study, Golgi protein 73 (GP73) of hepatocelluler carcinoma (HCC) cell lines with different metastatic potential was purified by antibody affinity chromatography and the purification result was confirmed by LC-MS/MS, providing more proof for GP73 as a biomarker for HCC. Purified GP73 was then analyzed using western blot and lectin blot to obtain its glycosylation level in HCC cell lines with different metastatic potential. We found that glycan of GP73 can be recognized by ConA, AAL, LCA and PHA-E lectins, while the LCA and PHA-E-reactive fraction of GP73 was increased along with the increasing metastatic potential of HCC cell lines. The glycan level of GP73 was different in HCC cell lines with different metastatic potential, and the content of fucose (recognized by LCA) and bisecting structure (recognized by PHA-E) increased in agreement with the metastatic potential.
[1] Marrero J A, Romano P R, Nikolaeva O, et al. GP73, a resident Golgi glycoprotein, is a novel serum marker for hepatocellular carcinoma[J]. Journal of Hepatology, 2005, 43(6): 1007-1012.
[2] Bachert C, Fimmel C, Linstedt A D. Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GP73 produces marker for hepatocellular carcinoma[J]. Traffic, 2007, 8(10): 1415-1423.
[3] Mao Y, Yang H, Xu H, et al. Golgi protein 73 (GOLPH2) is a valuable serum marker for hepatocellular carcinoma[J]. Gut, 59(12): 1687-1693.
[4] Drake R R, Schwegler E E, Malik G, et al. Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers[J]. Molecular of Cell Proteomics, 2006, 5(10): 1957-1967.
[5] Hu L, Li L, Xie H, et al. The Golgi localization of GOLPH2 (GP73/ GOLM1) is determined by the transmembrane and cytoplamic sequences[J]. PLoS One, 2011, 6(11): e28207.
[6] Norton P A, Comunale M A, Krakover J, et al. N-linked glycosylation of the liver cancer biomarker GP73[J]. Journal of Cell Biochemistry, 2008, 104(1): 136-149.
[7] Wang M, Long R E, Comunale M A, et al. Novel fucosylated biomarkers for the early detection of hepatocellular carcinoma[J]. Cancer Epidemiology Biomarkers Prevention, 2009, 18(6): 1914-1921.
[8] Block T M, Comunale M A, Lowman M, et al. Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(3): 779-784.
[9] Morelle W, Michalski J C, Analysis of protein glycosylation by mass spectrometry[J]. Nature Protocols, 2007, 2(7): 1585-1602.
[10] Marino K, Bones J, Kattla J J, et al. A systematic approach to protein glycosylation analysis: A path through the maze[J]. Nature Chemical Biology, 6(10): 713-723.
[11] Amos B, Lotan D, Lotan R. Increased fucosylation of high-molecularweight glycoproteins accompanies retinoic-acid-induced differentiation of F-9 embryonal carcinoma cells[J]. International Journal of Cancer, 1990, 46(1): 86-94.
[12] Nakagawa T, Miyoshi E, Yakushijin T, et al. Glycomic analysis of alpha-fetoprotein L3 in hepatoma cell lines and hepatocellular carcinoma patients[J]. Journal of Proteome Research, 2008, 7(6): 2222-2233.
[13] Yamashita K, Koide N, Endo T, et al. Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma[J]. The Journal of Biological Chemistry, 1989, 264(5): 2415-2423.
[14] Nishihara S, Iwasaki H, Kaneko M, et al. Alpha1, 3-fucosyltransferase 9 (FUT9, Fuc-TIX) preferentially fucosylates the distal GlcNAc residue of polylactosamine chain while the other four alpha1, 3FUT members preferentially fucosylate the inner GlcNAc residue[J]. FEBS Letters, 1999, 462(3): 289-294.
[15] Orntoft T F, Vestergaard E M, Clinical aspects of altered glycosylation of glycoproteins in cancer[J]. Electrophoresis, 1999, 20(2): 362-371.
[16] Kim Y J, Varki A, Perspectives on the significance of altered glycosylation of glycoproteins in cancer[J]. Glycoconjugate Journal, 1997, 14(5): 569-576.
[17] Miyoshi E, Ihara Y, Hayashi N, et al. Transfection of Nacetylglucosaminyltransferase Ⅲ gene suppresses expression of hepatitis B virus in a human hepatoma cell line, HB611[J]. The Journal of Biological Chemistry, 1995, 270(47): 28311-28315.
[18] Kang S K, Chung T W, Lee J Y, et al. The hepatitis B virus X protein inhibits secretion of apolipoprotein B by enhancing the expression of N-acetylglucosaminyltransferase Ⅲ[J]. The Journal of Biological Chemistry, 2004, 279(27): 28106-28112.
[19] 董芳, 肖凡, 魏红山. 平分型GlcNAc糖基化修饰的生物学功能[J]. 医 学分子生物学杂志, 2009, 6(2): 138-143. Dong fang, Xiao fan, Wei hongshan. Bisecting N-acetylglucosamine and its biological functions[J]. Journal of Medical Molecular Biology, 2009, 6(2): 138-143.