抗人ppGalNAc-T2单克隆抗体制备以及慢病毒介导ppGalNAc-T2基因表达对白血病细胞恶性表型的影响
|
摘要
目的:O-糖基化异常与多种疾病密切相关,ppGalNAc-Ts酶家族作为催化黏蛋白型O-聚糖合成的起始酶,在肿瘤中出现的ppGalNAc-Ts异常表达已经被认为是肿瘤特异聚糖结构产生的重要机制之一。有研究发现,与正常组织或细胞相比,ppGalNAc-T2在结肠癌、乳腺癌等肿瘤中存在异常表达。为了研究ppGalNAc-T2基因在表达及其功能,本实验制备特异性抗人ppGalNAc-T2单克隆抗体,并对该抗体的特性及应用进行分析。
方法:(1)抗原制备:利用PCR的方法从人293T细胞中克隆ppGalNAc-T2的编码区cDNA,构建pET-32a(+)-ppGalNAc-T2原核表达重组载体,载体经鉴定后转化至大肠杆菌BL21宿主菌,采用自动诱导方案表达ppGalNAc-T2融合蛋白;融合蛋白经凝胶纯化并鉴定后获得免疫用抗原;(2)杂交瘤及单克隆抗体制备:免疫BALB/c小鼠,利用细胞融合技术构建杂交瘤细胞,经间接ELISA和流式细胞术双筛选方法来获得稳定、高效分泌抗ppGalNAc-T2抗体的杂交瘤细胞株;然后采用小鼠体内腹水诱生法大量制备腹水型单抗;(3)单克隆抗体特性分析及其应用:测定单克隆抗体的亚类,然后将单克隆抗体用于间接ELISA,原核蛋白及真核蛋白的Western Blot分析,流式细胞术以及细胞免疫荧光分析。
结果:筛选出一株稳定分泌抗人ppGalNAc-T2单克隆抗体的杂交瘤细胞株,命名为LW-5F3,保藏号NO.C2010106;该单抗亚类为IgMκ;Western blot分析该单克隆抗体不仅识别原核表达的ppGalNAc-T2融合蛋白,还特异识别L929-ppGalNAc-T2转基因细胞表达的ppGalNAc-T2真核蛋白;流式细胞术分析检测到白血病Jurkat细胞和肝癌HepG2细胞均表达ppGalNAc-T2蛋白,并对肝癌HepG2细胞中ppGalNAc-T2蛋白表达情况进行了免疫荧光分析。
第二部分:慢病毒介导ppGalNAc-T2基因表达对白血病细胞恶性表型的影响
目的:白血病细胞株在经全反式维甲酸诱导分化过程中ppGalNAc-T2基因出现表达异常,而且有研究发现抑制O-糖基化可影响结肠癌和淋巴瘤等肿瘤的生长恶化。我们推测ppGalNAc-T2的表达及其控制合成的O-糖链在白血病中可能有着重要的作用。为此我们利用慢病毒转染手段,来研究ppGalNAc-T2基因表达对白血病Jurkat细胞恶性表型的影响,来探讨O-糖基化在白血病中的作用。
方法:(1)慢病毒过表达载体构建:利用PCR的方法从人293T细胞中克隆ppGalNAc-T2的编码区cDNA,构建慢病毒重组质粒venus-ppGalNAc-T2,将重组质粒及包装质粒共转染至293T细胞产生病毒颗粒,毒液经纯化并进行滴度测定;(2)慢病毒RNAi载体构建:设计并合成靶向沉默ppGalNAc-T2基因表达的shRNA及negative control,经退火后连接至慢病毒载体YH1上,病毒产生及滴度测定等操作同上;(3)白血病Jurkat细胞转染:将(1)和(2)所得慢病毒感染Jurkat细胞,然后进行经流式分选仪分选YFP阳性细胞,每个单细胞单独培养为单克隆细胞株,经流式细胞术,半定量RT-PCR和Western blot鉴定后,获得Jurkat细胞ppGalNAc-T2过表达及RNAi模型;(4)实验分析ppGalNAc-T2表达改变后,Jurkat细胞增殖、粘附以及TransWell迁移能力的改变。
结果:成功构建了重组慢病毒ppGalNAc-T2过表达载体和慢病毒RNAi载体,感染Jurkat细胞经流式细胞仪分选,半定量RT-PCR和Western blot实验鉴定获得了稳定转染Jurkat细胞株;MTT实验,细胞粘附实验和Transwell细胞迁移实验分析表明ppGalNAc-T2过表达可促进Jurkat细胞的增殖,粘附及细胞迁移能力;当ppGalNAc-T2表达被RNAi沉默后,Jurkat细胞的增殖,粘附和迁移能力降低。
Objective: UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminy- ltransferases (ppGalNAc-Ts) regulate the initial key step of mucin O-glycosylation. ppGalNAc-T2 as a key member of ppGalNAc-T family, was recently described abbrant expression in oral squamous cell carcinoma, colorectal and breast carcinoma. In order to gain further insight into the role of ppGalNAc-T2, we decide to produce the anti-human ppGalNAc-T2 monoclonal antibody.
Methods: (1) The cDNA were obtained from the human 293T cells, the full length of human ppGalNAc-T2 gene were amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a(+) and transformed into E.coli BL21(DE3). After induced by auto-induction, the recombinant protein was expressed and purified using protein electroelution purification. (2) Immunized BALB/c mice with the recombinant human ppGalNAc-T2 protein, the MAb against ppGalNAc-T2 was prepared using hybridoma technique. The positive hybridoma was selected by ELISA and Flow Cytometry. Ascites MAb were obtained after inoculation of isolated hybridoma into BALB /c mice. (3) The MAb was used for ELISA, Western Blot(fusion protein and eukaryotic protein from transgenic L929-ppGalNAc-T2 cells), Flow Cytometry(Jurkat,HepG2) and immunofluorescence(HepG2,LSC).
Results: Hybridoma LW-5F3 cell line was obtain, It’s IgMκisotype, mAb can specifically recognize human ppGalNAc-T2 protein in both fusion and native formats by Western blot, Flow cytometry and immunofluorescent staining.
PartⅡ. The Effect of ppGalNAc-T2 Gene Expression Mediated by Lentivirus on the Malignant Phenotype of Jurkat Cell Line
Objective: ppGalNAc-T2 expression pattern has been described in many human tumors, and our lab has demonstrated that it’s expression was also changed during 1,25(OH)2D3 induced differentiation in several leukemia cell lines. Based on these studies, we construct the recombinant lentivirus, in ordor to study the effection of ppGalNAc-T2 expression on Jurkat malignant phenotypes in vitro.
Methods: (1) The cDNA were obtained from the human 293T cells, the full length of human ppGalNAc-T2 gene were amplified by RT-PCR. Then it was recombined into the lentiviral vector pRRL venus, After enzyme digestion and sequencing confirmation, each of the recombinant vectors and packaging vectors were cotransducted into 293T cells, the recombinant lentivirus were packaged and then purified. (2) The RNAi and negative control shRNA were designed and synthesisd, then were inserted into lentivirus vector YH1. After enzyme digestion and sequencing confirmation, each of the recombinant vectors and packaging vectors were cotransducted into 293T cells, and the recombinant lentivirus were packaged and then purified. (3) Jurkat cells were infected by purified lentivirus, and then sorted the YFP positive cells into 96-well plate(1cell/well) by High-speed cell sorter (Beckman Coulter), the ppGalNAc-T2 mRNA and protein expression levels were analyzed using RT-PCR and Western Blotting. (4) The malignant phenotype of Jurkat cells were analyzed using MTT, cell aggregation and Transwell assays.
Results: Lentiviral vector-based over-expression and siRNA expression plasmids against human ppGalNAc-T2 gene have been successfully constructed and identified by DNA sequencing. And stable trangenic Jurkat cells (over-expression and RNAi) were obtained. Over-expression of ppGalNAc-T2 could promote cell proliferation, aggregation and migration of acute lymphocytic leukemia Jurkat cells.
方法:(1)抗原制备:利用PCR的方法从人293T细胞中克隆ppGalNAc-T2的编码区cDNA,构建pET-32a(+)-ppGalNAc-T2原核表达重组载体,载体经鉴定后转化至大肠杆菌BL21宿主菌,采用自动诱导方案表达ppGalNAc-T2融合蛋白;融合蛋白经凝胶纯化并鉴定后获得免疫用抗原;(2)杂交瘤及单克隆抗体制备:免疫BALB/c小鼠,利用细胞融合技术构建杂交瘤细胞,经间接ELISA和流式细胞术双筛选方法来获得稳定、高效分泌抗ppGalNAc-T2抗体的杂交瘤细胞株;然后采用小鼠体内腹水诱生法大量制备腹水型单抗;(3)单克隆抗体特性分析及其应用:测定单克隆抗体的亚类,然后将单克隆抗体用于间接ELISA,原核蛋白及真核蛋白的Western Blot分析,流式细胞术以及细胞免疫荧光分析。
结果:筛选出一株稳定分泌抗人ppGalNAc-T2单克隆抗体的杂交瘤细胞株,命名为LW-5F3,保藏号NO.C2010106;该单抗亚类为IgMκ;Western blot分析该单克隆抗体不仅识别原核表达的ppGalNAc-T2融合蛋白,还特异识别L929-ppGalNAc-T2转基因细胞表达的ppGalNAc-T2真核蛋白;流式细胞术分析检测到白血病Jurkat细胞和肝癌HepG2细胞均表达ppGalNAc-T2蛋白,并对肝癌HepG2细胞中ppGalNAc-T2蛋白表达情况进行了免疫荧光分析。
第二部分:慢病毒介导ppGalNAc-T2基因表达对白血病细胞恶性表型的影响
目的:白血病细胞株在经全反式维甲酸诱导分化过程中ppGalNAc-T2基因出现表达异常,而且有研究发现抑制O-糖基化可影响结肠癌和淋巴瘤等肿瘤的生长恶化。我们推测ppGalNAc-T2的表达及其控制合成的O-糖链在白血病中可能有着重要的作用。为此我们利用慢病毒转染手段,来研究ppGalNAc-T2基因表达对白血病Jurkat细胞恶性表型的影响,来探讨O-糖基化在白血病中的作用。
方法:(1)慢病毒过表达载体构建:利用PCR的方法从人293T细胞中克隆ppGalNAc-T2的编码区cDNA,构建慢病毒重组质粒venus-ppGalNAc-T2,将重组质粒及包装质粒共转染至293T细胞产生病毒颗粒,毒液经纯化并进行滴度测定;(2)慢病毒RNAi载体构建:设计并合成靶向沉默ppGalNAc-T2基因表达的shRNA及negative control,经退火后连接至慢病毒载体YH1上,病毒产生及滴度测定等操作同上;(3)白血病Jurkat细胞转染:将(1)和(2)所得慢病毒感染Jurkat细胞,然后进行经流式分选仪分选YFP阳性细胞,每个单细胞单独培养为单克隆细胞株,经流式细胞术,半定量RT-PCR和Western blot鉴定后,获得Jurkat细胞ppGalNAc-T2过表达及RNAi模型;(4)实验分析ppGalNAc-T2表达改变后,Jurkat细胞增殖、粘附以及TransWell迁移能力的改变。
结果:成功构建了重组慢病毒ppGalNAc-T2过表达载体和慢病毒RNAi载体,感染Jurkat细胞经流式细胞仪分选,半定量RT-PCR和Western blot实验鉴定获得了稳定转染Jurkat细胞株;MTT实验,细胞粘附实验和Transwell细胞迁移实验分析表明ppGalNAc-T2过表达可促进Jurkat细胞的增殖,粘附及细胞迁移能力;当ppGalNAc-T2表达被RNAi沉默后,Jurkat细胞的增殖,粘附和迁移能力降低。
Objective: UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminy- ltransferases (ppGalNAc-Ts) regulate the initial key step of mucin O-glycosylation. ppGalNAc-T2 as a key member of ppGalNAc-T family, was recently described abbrant expression in oral squamous cell carcinoma, colorectal and breast carcinoma. In order to gain further insight into the role of ppGalNAc-T2, we decide to produce the anti-human ppGalNAc-T2 monoclonal antibody.
Methods: (1) The cDNA were obtained from the human 293T cells, the full length of human ppGalNAc-T2 gene were amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a(+) and transformed into E.coli BL21(DE3). After induced by auto-induction, the recombinant protein was expressed and purified using protein electroelution purification. (2) Immunized BALB/c mice with the recombinant human ppGalNAc-T2 protein, the MAb against ppGalNAc-T2 was prepared using hybridoma technique. The positive hybridoma was selected by ELISA and Flow Cytometry. Ascites MAb were obtained after inoculation of isolated hybridoma into BALB /c mice. (3) The MAb was used for ELISA, Western Blot(fusion protein and eukaryotic protein from transgenic L929-ppGalNAc-T2 cells), Flow Cytometry(Jurkat,HepG2) and immunofluorescence(HepG2,LSC).
Results: Hybridoma LW-5F3 cell line was obtain, It’s IgMκisotype, mAb can specifically recognize human ppGalNAc-T2 protein in both fusion and native formats by Western blot, Flow cytometry and immunofluorescent staining.
PartⅡ. The Effect of ppGalNAc-T2 Gene Expression Mediated by Lentivirus on the Malignant Phenotype of Jurkat Cell Line
Objective: ppGalNAc-T2 expression pattern has been described in many human tumors, and our lab has demonstrated that it’s expression was also changed during 1,25(OH)2D3 induced differentiation in several leukemia cell lines. Based on these studies, we construct the recombinant lentivirus, in ordor to study the effection of ppGalNAc-T2 expression on Jurkat malignant phenotypes in vitro.
Methods: (1) The cDNA were obtained from the human 293T cells, the full length of human ppGalNAc-T2 gene were amplified by RT-PCR. Then it was recombined into the lentiviral vector pRRL venus, After enzyme digestion and sequencing confirmation, each of the recombinant vectors and packaging vectors were cotransducted into 293T cells, the recombinant lentivirus were packaged and then purified. (2) The RNAi and negative control shRNA were designed and synthesisd, then were inserted into lentivirus vector YH1. After enzyme digestion and sequencing confirmation, each of the recombinant vectors and packaging vectors were cotransducted into 293T cells, and the recombinant lentivirus were packaged and then purified. (3) Jurkat cells were infected by purified lentivirus, and then sorted the YFP positive cells into 96-well plate(1cell/well) by High-speed cell sorter (Beckman Coulter), the ppGalNAc-T2 mRNA and protein expression levels were analyzed using RT-PCR and Western Blotting. (4) The malignant phenotype of Jurkat cells were analyzed using MTT, cell aggregation and Transwell assays.
Results: Lentiviral vector-based over-expression and siRNA expression plasmids against human ppGalNAc-T2 gene have been successfully constructed and identified by DNA sequencing. And stable trangenic Jurkat cells (over-expression and RNAi) were obtained. Over-expression of ppGalNAc-T2 could promote cell proliferation, aggregation and migration of acute lymphocytic leukemia Jurkat cells.
引文
1. Ohtsubo K, Marth J D. Glycosylation in cellular mechanisms of health and disease. Cell. 2006, 126(5): 855～867
2. Springer G F. T and Tn, general carcinoma autoantigens. Science. 1984, 224(4654):1198～206
3. Meezan E, Wu H C, Black P H, et al. Comparative studies on carbohydrate- containingmembrane components of normal and virus-transformed mouse fibroblasts. Separation of glycoproteins and glycopeptides by sephadex chromatography. Biochemistry. 1969, 8:2518～2524
4. Zachary Shriver, S. Raguram & Ram Sasisekharan. Glycomics: a pathway to a class of new and improved therapeutics. Nat Rev Drug Discovery. 2004,3:863～873
5. Senitiroh Hakomori. Glycosylation defining cancer malignancy: New wine in an old bottle. Proc Natl Acad Sci USA. 2002, 99 (16):10231～10233
6. Marth J D, Grewal P K. Mammalian glycosylation in immunity. Nat Rev Immunol. 2008, 8(11): 874～87
7. Dube D H, Bertozzi C R. Glycans in cancer and inflammation-potential for therapeutics and diagnostics. Nat Rev Drug Discovery. 2005, 4(6): 477～488
8. Hollingsworth M A, Swanson B J. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004, 4(1): 45～60
9. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochimica Biophysica Acta. 1999, 1473(1):67～95
10. Torben F Orntoft and Else Marie Vestergaard. Clinical aspects of altered glycosylation of glycoproteins in cancer. Electrophoresis.1999,20(2):362～371
11. Ten Hagen K G, Fritz T A and Tabak L A. All in the family: the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases. Glycobiology. 2003, 13(1): 1R～16R
12. Peng C, Togayachi A, Kwon Y D, et al. Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile. Biochem Biophys Res Commun. 2010,402 (4): 680～686
13. Chen Wu, Xiaodan Guo, Weina Wang, et al. RN-Acetylgalactosaminy- ltransferase-14 as a potential biomarker for breast cancer by immunohistochemistry. BMC Cancer. 2010, 10:123
14. Potapenko I O, Haakensen V D, Ludersc T, et al. Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 2010, 4(2):9 8～118
15. Kentaro Kato, Charlotte Jeanneau, Mads Agervig Tarp, et al. Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis: SECRETION OF FIBROBLAST GROWTH FACTOR 23 REQUIRES O-GLYCOSYLATION. J Biol Chem. 2006 281: 18370～18377
16. Klaus W Wagner, Elizabeth A Punnoose, Thomas Januario, et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med. 2007,13(9):1070～1077
17. Herr P, Korniychuk G, Yamamoto Y, et al. Regulation of TGF-βsignalling by N-acetylgalactosaminyltransferase-like 1. Development. 2008,135:1813～1822
18. Giuseppe Merla, Catherine Ucla, Michel Guipponi, et al. Identification of additional transcripts in the Williams-Beuren syndrome critical region. Human Genetics. 2002, 110:429～438
19. Naosuke Nakamura, Shinya Toba, Mitsuharu Hirai, et al. Cloning and Expression of a Brain-Specific Putative UDP-GalNAc: PolypeptideN-Acetylgalactosaminy- ltransferase Gene. Biological & Pharmaceutical Bulletin. 2005, 28:429～433
20. Andrew D. Simmons, Maurice M. Musy, Carla S. Lopes, et al. A Direct Interaction Between EXT Proteins and Glycosyltransferases is Defective in Hereditary Multiple Exostoses. Hum Mol Genet.1999, 8(12): 2155～2164
21. Jian-Ming Guo, Hui-Li Chen, Guo-Min Wang, et al. Expression of UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase-12 in Gastric and Colonic Cancer Cell Lines and in Human Colorectal Cancer. Oncology. 2004, 67:271～276
22. Laura D Wood, D Williams Parsons, Sian Jones, et al. The Genomic Landscapes of Human Breast and Colorectal Cancers. Science. 2007, 318:1108～1113
23. Kishore Guda, Helen Moinova, Jian He, et al. Inactivating germ-line and somatic mutations in polypeptide N-acetylgalactosaminyltransferase 12 in human colon cancers. Proc Natl Acad Sci USA. 2009, 106(31):12921～12925
24. Cristen J Willer, Serena Sanna, Anne U Jackson, et al. Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nat Genet. 2008, 40(2):161～169
25. Sekar Kathiresan, Olle Melander, Candace Guiducci, et al. Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet. 2008, 40(2):189～197
26. Tanya M Teslovich, Kiran Musunuru, Albert V Smith, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010, 466:707～713
27. Tarp MA, Clausen H. Mucin-type O-glycosylation and its potential use in drug and vaccine development. Biochim Biophys Acta. 2008 1780(3):546～563
28. Patsos G, Hebbe-Viton V, Robbe-Masselot C, et al. O-Glycan inhibitors generate aryl-glycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines. Glycobiology. 2009, 19(4):382～398
29. Takuhiro Kohsaki, Isao Nishimori, Hirofumi Nakayama, et al. Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol. 2000, 35:840～848
30. Qiu H, Guo X H, Mo J H, et al. Expressions of polypeptide: N-acetylgalact- osaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation. Glycoconj J. 2006, 23(7-8): 575～584
31. Keita Yamada, Mitsuhiro Kinoshita, Takao Hayakawa, et al. Comparative Studies on the Structural Features of O-Glycans between Leukemia and Epithelial Cell Lines. Journal of Proteome Research. 2009, 8: 521～537
32. Naldini L, Bl?mer U, Gallay P, et al. In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector. Science. 1996, 272(5259):263～267
33. Hannon G J. RNA interference. Nature. 2002, 418(6894):244～251
34. Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003, 9 (4) :493～501
35. Sumimoto H and Kawakami Y. Lentiviral vector-mediated RNAi and its use for cancer research. Future Oncol. 2007,3(6):655～664
1. Brockhausen I.Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Report. 2006, 7:599～604
2. Peng C, Togayachi A, Kwon YD, et al. Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile. Biochem Biophys Res Commun. 2010, 402:680～686
3. Ten Hagen KG, Fritz TA, Tabak LA. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology. 2003, 13:1R～16R
4. Mandel U, Hassan H, Therkildsen MH, et al. Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas:immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family. Glycobiology. 1999, 9:43～52
5. Kohsaki T, Nishimori I, Nakayama H, et al. Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol. 2000, 35:840～848
6. Potapenko IO, Haakensen VD, Luders T, et al. Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 2010, 4:98～118
7. Galfre G and Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol.1981,73:3
8. Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005, 41:207～234
1. Springer G F. T and Tn, general carcinoma autoantigens. Science. 1984, 224(4654):1198～206
2. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochimica Biophysica Acta. 1999. 1473(1):67～95
3. Hollingsworth M A, Swanson B J. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004, 4(1): 45～60
4. Brockhausen I. Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO reports. 2006, 7(6):599～604
5. Hector F Valenzuela, Karen E Pace, Paula V Cabrera, et al. O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1. Cancer Res. 2007, 67: (13):6155～6162
6. E Rajpert-De Meyts, S N Poll, I Goukasian, et al. Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation. Virchows Arch. 2007, 451:805～814
7. Liying Chen, Jonas Sundback, Sigvard Olofsson, et al. Interference with O-glycosylation in RMA lymphoma cells leads to a reduced in vivo growth of the tumor. Int J Cancer. 2006, 119:1495～1500
8. Georgios Patsos, Virginie Hebbe-Viton, Catherine Robbe-Masselot, et al. O-Glycan inhibitors generate aryl-glycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines. Glycobiology. 2009,19(4): 382～398
9. Hanisch F G, Reis C A, Clausen H, et al. Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides. Glycobiology. 2001, 11(9):731～740
10. Takuhiro Kohsaki, Isao Nishimori, Hirofumi Nakayama, et al. Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol. 2000, 35:840～848
11. Potapenko I O, Haakensen V D, Ludersc T, et al. Glycan gene expressionsignatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 2010, 4(2): 98～118
12. Qiu H, Guo X H, Mo J H, et al. Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation. Glycoconj J. 2006, 23(7-8):575～584
13. Schneider U, Schwenk H, Bornkamm G. Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer. 1977, 19 (5): 621～626
14. Yamada K, Kinoshita M, Hayakawa T, et al. Comparative Studies on the Structural Features of O-Glycans between Leukemia and Epithelial Cell Lines. Journal of Proteome Research. 2009, 8(2):521～537
15. Hannon G J. RNA interference. Nature. 2002, 418(6894):244～251
16. Naldini L, Bl?mer U, Gallay P, et al. In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector. Science. 1996, 272(5259): 263～267
17. Herold M J, van den Brandt J, Seibler J, et al. Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc Natl Acad Sci USA. 2008, 105(47): 18507～18512
18. Hitomi Tsuiji, Seiichi Takasaki, Michiie Sakamoto, et al. Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinoma-associated antigen, and facilitates cell-cell adhesion. Glycobiology. 2003, 13(7):521～527
19. Phillips ML, Nudelman E, Gaeta FCA, et al. ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex. Science. 1990, 250: 1130～1132
20. Takada A, Ohmori K, Takahashi N, et al. Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. Biochem Biophys Res Commun. 1991; 179: 713～719
21. Takada A, Ohmori K, Yoneda T, et al. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res. 1993; 53: 354～361
22. Kannagi R. Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer. Glycoconj J. 1997; 14: 577～584
23. Ito K, Ye CL, Hibi K, Mitsuoka C, et al. Paired tumor marker of soluble E-selectin and its ligand sialyl Lewis A in colorectal cancer. J Gastroenterol. 2001; 36: 823～829
24. Reiji Kannagi, Mineko Izawa, Tetsufumi Koike, et al. Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis. Cancer Sci. 2004,95(5):377～384
1. Turnbull J E, Field A R. Emerging glycomics technologies. Nature chemical biology, 2007, 3(2): 74～77
2. Ohtsubo K, Marth J D. Glycosylation in cellular mechanisms of health and disease. Cell, 2006, 126(5): 855～867
3. Marth J D, Grewal P K. Mammalian glycosylation in immunity. Nat Rev Immunol, 2008, 8(11): 874-87
4. Haltiwanger R S, Lowe J B. Role of glycosylation in development. Annu Rev Biochem, 2004, 73: 491～537
5. Wopereis S, Lefeber D J, Morava E, et al. Mechanisms in protein O-glycan biosynthesis and clinical and molecular aspects of protein O-glycan biosynthesis defects: A Review. Clinical Chemistry, 2006, 52(4): 574～600
6. Hollingsworth M A, Swanson B J. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer, 2004, 4(1): 45～60
7. Ju T, Brewer K, D’Souza A, et al. Cloning and expression of human core 1β1,3-galactosyltransferase. J Biol Chem, 2002, 277(1): 178～186
8. Narimatsu Y, Ikehara Y, Iwasaki H, et al. Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc. Biochem Biophys Res Commun, 2008, 366(1): 199～205
9. Ju T, Cummings R D. Protein glycosylation: chaperone mutation in Tn syndrome. Nature, 2005, 437(7063): 1252
10. Schietinger A, Philip M, Yoshida B A, et al. A mutant chaperone converts a wild-type protein into a tumor-specific antigen. Science, 2006, 314(5797): 304～308
11. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochim Biophys Acta, 1999, 1473(1): 67～95
12. Fukuda M. Roles of mucin-type O-glycans synthesized by core2beta1,6-N-acetylglucosaminyltransferase. Methods Enzymol, 2006, 416: 332～346
13. Ten Hagen K G, Fritz T A, Tabak L A. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology,2003, 13(1): 1R～16R
14. Homa F L, Hollander T, Lehman D J, et al. Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminy- ltransferase. J Biol Chem, 1993, 268(17): 12609～12616
15. Hagen F K, VanWuyckhuyse B, Tabak L A. Purification, cloning and expression of a bovine UDP-GalNAc:polypeptide N-Acetylgalactosaminyltransferase. J Biol Chem, 1993, 268(25): 18960～18965
16. Hagen F K, Gregoire C A, Tabak L A. Cloning and sequence homology of a rat UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Glycoconj J, 1995, 12(6): 901～909
17. White T, Bennett E P, Takio K, et al. Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem, 1995, 270(41): 24156～24165
18. Hagen F K, Ten Hagen K G, Beres T M, et al. cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminy- ltransferase. J Biol Chem, 1997, 272(21): 13843～13848
19. Yoshida A, Hara T, Ikenaga H, et al. Cloning and expression of a porcine UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase. Glycoconj J, 1995, 12(6): 824～828
20. Bennett E P, Hassan H, Clausen H. cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3. J Biol Chem, 1996, 271(29): 17006～17012
21. Zara J, Hagen F K, Ten Hagen K G, et al. Cloning and expression of mouse UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase-T3. Biochem Biophys Res Comm, 1996, 228(1): 38～44
22. Bennett E P, Hassan H, Mandel U, et al. Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine: polypeptide Nacetylgalactosaminyltransferase that complements other GalNActransferases in complete O-glycosylation of the MUC1 tandem repeat. J Biol Chem, 1998, 273(46): 30472～30481
23. Ten Hagen K G, Hagen F K, Balys M M, et al. Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc: polypeptide N-acetylgala ctosaminyltransferase family. J Biol Chem, 1998, 273(42): 27749～27754
24. Bennett E P, Hassan H, Mandel U, et al. Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine: polypeptide Nacetylgalactosaminyltransferase-T3, designated GalNAc-T6. J Biol Chem, 1999, 274(36): 25362～25370
25. Ten Hagen K G, Tetaert D, Hagen F K, et al. Characterization of a UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases that displays glycopeptide N-acetylgalactosaminyl transferases activity. J Biol Chem, 1999, 274(39): 27867～27874
26. Bennett E P, Hassan H, Hollingsworth M A, et al. A novel human UDP-N-acetyl-D-galactosamine:polypeptide Nacetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates. FEBS Letters, 1999, 460(2): 226～230
27. White K E, Lorenz B, Evans W E, et al. Molecular cloning of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene, 2000, 246(1-2): 347～356
28. Toba S, Tenno M, Konishi M, et al. Brain-specific expression of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9). Biochim Biophys Acta, 2000, 1493(1-2): 264～268
29. Zhang Y, Iwasaki H, Wang H, et al. Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminy- ltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen. J Biol Chem, 2003, 278(1): 573～584
30. Ten Hagen K G., Bedi G S, Tetaert D, et al. Cloning and characterization of a ninth member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases family, ppGaNTase-T9. J Biol Chem, 2001, 276(20):17395～17404
31. Cheng L M, Tachibana K, Zhang Y, et al. Characterization of a novel humanUDP-GalNAc transferase, pp-GalNAc-T10. FEBS Lett, 2002, 531(2): 115～121
32. Schwientek T, Bennett E P, Flores C, et al. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminy- ltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila. J Biol Chem, 2002, 277(25): 22623～22638
33. Guo J M, Zhang Y, Cheng L, et al. Molecular cloning and characterization of a novel member of the UDP-GalNAc: Polypeptide N-acetylgalactosaminy- ltransferase family, pp-GalNAc-T12. FEBS Letters, 2002, 524(1-3): 211～218
34. Hennet T, Hagen F K, Tabak L A, et al. T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination. Proc Natl Acad Sci USA, 1995, 92(26): 12070～12074
35. Wang H, Tachibana K, Zhang Y, et al. Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase,pp-GalNAc-T14. Biochem Biophys Res Commun, 2003, 300(3): 738～744
36. Cheng L, Tachibana K, Iwasaki H, et al. Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15. FEBS Lett, 2004, 566(1-3): 17～24
37. Wang J, Shao D, Xie C, et al. The research of a novel UDP-N-acetyl-α-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase like gene. 21st IUBMB and 12st FAOBMB International Congress of Biochemistry and Molecular Biology, Shanghai, August 2-7, 2009[C]. pp195: E-2-020
38. Peng C, Togayachi A, Kwon Y D, et al. Cloning and characterization of a novel human UDP-GalNAc transferase, ppGalNAc-T20. 21st IUBMB and 12st FAOBMB International Congress of Biochemistry and Molecular Biology, Shanghai, August 2-7, 2009[C]. pp195: E-2-022
39. R(?)ttger S, White J, Wandall H H, et al. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J Cell Sci, 1998, 111(Pt1): 45～60
40. Zhou Y H, Hang S Y, Qiu H, et al. UDP-GalNAc: polypeptideα-N-acetygalactosaminyltransferase 2 localized on both cis and trans side of Golgi stacks in SGC7901 cells. Progress in Biochemistry and Biophysics, 2009, 36(1): 49～57
41. Wandall H H, Irazoqui F, Tarp M A, et al. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. Glycobiology, 2007, 17(4): 374～387
42. Fritz T A, Hurley J H, Trinh L B, et al. The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminy- ltransferase-T1. Proc Natl Acad Sci USA, 2004, 101(43), 15307～15312
43. Fritz T A, Raman J, Tabak L A. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminy- ltransferase-2. J Biol Chem, 2006, 281(13): 8613～8619
44. Kubota T, Shiba T, Sugioka S, et al. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminy- ltransferase (pp-GalNAc-T10). J Mol Biol, 2006, 359(3): 708～727
45. Marchler-Bauer A, Bryant S H. CD-Search: protein domain annotations on the fly. Nucleic Acids Res, 2004, 32: W327～331
46. Berois N, Mazal D, Ubillos L, et al. UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker. J Histochem Cytochem, 2006, 54(3): 317～328
47. Inoue T, Eguchi T, Oda Y, et al. Expression of GalNAc-T3 and its relationships with clinicopathological factors in 61 extrahepatic bile duct carcinomas analyzed using stepwise sections - special reference to its association with lymph node metastases-. Mod Pathol, 2007, 20(2): 267～276
48. Gomes J, Marcos N T, Berois N, et al. Expression of UDP-N-acetyl-D-galactosamine: Polypeptide N-acetylgalactosaminyltransferase-6 in gastric mucosa, intestinal metaplasia, and gastric carcinoma, Journal of Histochem Cytochem, 2009, 57(1): 79～86
49. Guo J M, Chen H L, Wang G M, et al. Expression of UDP-GalNAc: Polypeptide N-Acetylgalactosaminyl transferase-12 in gastric and colonic cancer cell lines and in human colorectal cancer. Oncology, 2004, 67(3-4): 271～276
50. Qiu H, Guo X H, Mo J H , et al. Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during1,25-dihydroxyvitamin D3 induced differentiation. Glycoconj J, 2006, 23(7-8): 575～584
51. Shimodaira K, Nakayama J, Nakamura N, et al. Carcinoma-associated expression of core 2β-1,6-Nacetylglucosaminyltransferase gene in human colorectal cancer: role of O-glycans in tumor progression. Cancer Res, 1997, 57(23): 5201～5206
52. Brockhausen I. Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO reports, 2006,7(6): 599-604
53. Wu L H, Hu P, Wu W, et al. Fucosylated glycans associated with development and metastasis of hepatocellular carcinoma cells. Progress in Biochemistry and Biophysics, 2009, 36(1): 33～41
54. Kanoh A, Takeuchi H, Kato K, et al. Interleukin-4 induces specific pp-GalNAc-T expression and alterations in mucin O-glycosylation in colonic epithelial cells. Biochimica et Biophysica Acta, 2008, 1780(3): 577～584
55. Higai K, Miyazaki N, Azuma Y, et al. Interleukin-1βinduces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene. FEBS Letters, 2006, 580(26): 6069～6075
56. Wu Y M, Nowack D D, Omenn G S, et al. Mucin glycosylation is altered by pro-inflammatory signaling in pancreatic-cancer cells. Journal of Proteome Research, 2009, 8(4):1876～1886
57. van Kooyk Y, Rabinovich G A. Protein-glycan interactions in the control of innate and adaptive immune responses. Nature Immunology, 2008, 9(6): 593-601
58. Tsuji H, Takasaki S, Sakamoto M, et al. Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinomaassociated antigen, and facilitates cell-cell adhesion. Glycobiology, 2003, 13(7): 521～527
59. Bao X F, Kobayashi M, Hatakeyama S, et al. Tumor suppressor function of laminin-bindingα-dystroglycan requires a distinctβ3-Nacetylglucosaminy- ltransferase. Proc Natl Acad Sci USA, 2009, 106(29):12109～12114
60.朱江龙, Danishefsky S J.前列腺癌和乳腺癌的免疫治疗新进展.科学通报, 2008, 53 (17): 2126
2. Springer G F. T and Tn, general carcinoma autoantigens. Science. 1984, 224(4654):1198～206
3. Meezan E, Wu H C, Black P H, et al. Comparative studies on carbohydrate- containingmembrane components of normal and virus-transformed mouse fibroblasts. Separation of glycoproteins and glycopeptides by sephadex chromatography. Biochemistry. 1969, 8:2518～2524
4. Zachary Shriver, S. Raguram & Ram Sasisekharan. Glycomics: a pathway to a class of new and improved therapeutics. Nat Rev Drug Discovery. 2004,3:863～873
5. Senitiroh Hakomori. Glycosylation defining cancer malignancy: New wine in an old bottle. Proc Natl Acad Sci USA. 2002, 99 (16):10231～10233
6. Marth J D, Grewal P K. Mammalian glycosylation in immunity. Nat Rev Immunol. 2008, 8(11): 874～87
7. Dube D H, Bertozzi C R. Glycans in cancer and inflammation-potential for therapeutics and diagnostics. Nat Rev Drug Discovery. 2005, 4(6): 477～488
8. Hollingsworth M A, Swanson B J. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004, 4(1): 45～60
9. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochimica Biophysica Acta. 1999, 1473(1):67～95
10. Torben F Orntoft and Else Marie Vestergaard. Clinical aspects of altered glycosylation of glycoproteins in cancer. Electrophoresis.1999,20(2):362～371
11. Ten Hagen K G, Fritz T A and Tabak L A. All in the family: the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases. Glycobiology. 2003, 13(1): 1R～16R
12. Peng C, Togayachi A, Kwon Y D, et al. Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile. Biochem Biophys Res Commun. 2010,402 (4): 680～686
13. Chen Wu, Xiaodan Guo, Weina Wang, et al. RN-Acetylgalactosaminy- ltransferase-14 as a potential biomarker for breast cancer by immunohistochemistry. BMC Cancer. 2010, 10:123
14. Potapenko I O, Haakensen V D, Ludersc T, et al. Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 2010, 4(2):9 8～118
15. Kentaro Kato, Charlotte Jeanneau, Mads Agervig Tarp, et al. Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis: SECRETION OF FIBROBLAST GROWTH FACTOR 23 REQUIRES O-GLYCOSYLATION. J Biol Chem. 2006 281: 18370～18377
16. Klaus W Wagner, Elizabeth A Punnoose, Thomas Januario, et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med. 2007,13(9):1070～1077
17. Herr P, Korniychuk G, Yamamoto Y, et al. Regulation of TGF-βsignalling by N-acetylgalactosaminyltransferase-like 1. Development. 2008,135:1813～1822
18. Giuseppe Merla, Catherine Ucla, Michel Guipponi, et al. Identification of additional transcripts in the Williams-Beuren syndrome critical region. Human Genetics. 2002, 110:429～438
19. Naosuke Nakamura, Shinya Toba, Mitsuharu Hirai, et al. Cloning and Expression of a Brain-Specific Putative UDP-GalNAc: PolypeptideN-Acetylgalactosaminy- ltransferase Gene. Biological & Pharmaceutical Bulletin. 2005, 28:429～433
20. Andrew D. Simmons, Maurice M. Musy, Carla S. Lopes, et al. A Direct Interaction Between EXT Proteins and Glycosyltransferases is Defective in Hereditary Multiple Exostoses. Hum Mol Genet.1999, 8(12): 2155～2164
21. Jian-Ming Guo, Hui-Li Chen, Guo-Min Wang, et al. Expression of UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase-12 in Gastric and Colonic Cancer Cell Lines and in Human Colorectal Cancer. Oncology. 2004, 67:271～276
22. Laura D Wood, D Williams Parsons, Sian Jones, et al. The Genomic Landscapes of Human Breast and Colorectal Cancers. Science. 2007, 318:1108～1113
23. Kishore Guda, Helen Moinova, Jian He, et al. Inactivating germ-line and somatic mutations in polypeptide N-acetylgalactosaminyltransferase 12 in human colon cancers. Proc Natl Acad Sci USA. 2009, 106(31):12921～12925
24. Cristen J Willer, Serena Sanna, Anne U Jackson, et al. Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nat Genet. 2008, 40(2):161～169
25. Sekar Kathiresan, Olle Melander, Candace Guiducci, et al. Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet. 2008, 40(2):189～197
26. Tanya M Teslovich, Kiran Musunuru, Albert V Smith, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature. 2010, 466:707～713
27. Tarp MA, Clausen H. Mucin-type O-glycosylation and its potential use in drug and vaccine development. Biochim Biophys Acta. 2008 1780(3):546～563
28. Patsos G, Hebbe-Viton V, Robbe-Masselot C, et al. O-Glycan inhibitors generate aryl-glycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines. Glycobiology. 2009, 19(4):382～398
29. Takuhiro Kohsaki, Isao Nishimori, Hirofumi Nakayama, et al. Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol. 2000, 35:840～848
30. Qiu H, Guo X H, Mo J H, et al. Expressions of polypeptide: N-acetylgalact- osaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation. Glycoconj J. 2006, 23(7-8): 575～584
31. Keita Yamada, Mitsuhiro Kinoshita, Takao Hayakawa, et al. Comparative Studies on the Structural Features of O-Glycans between Leukemia and Epithelial Cell Lines. Journal of Proteome Research. 2009, 8: 521～537
32. Naldini L, Bl?mer U, Gallay P, et al. In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector. Science. 1996, 272(5259):263～267
33. Hannon G J. RNA interference. Nature. 2002, 418(6894):244～251
34. Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003, 9 (4) :493～501
35. Sumimoto H and Kawakami Y. Lentiviral vector-mediated RNAi and its use for cancer research. Future Oncol. 2007,3(6):655～664
1. Brockhausen I.Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO Report. 2006, 7:599～604
2. Peng C, Togayachi A, Kwon YD, et al. Identification of a novel human UDP-GalNAc transferase with unique catalytic activity and expression profile. Biochem Biophys Res Commun. 2010, 402:680～686
3. Ten Hagen KG, Fritz TA, Tabak LA. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology. 2003, 13:1R～16R
4. Mandel U, Hassan H, Therkildsen MH, et al. Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas:immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family. Glycobiology. 1999, 9:43～52
5. Kohsaki T, Nishimori I, Nakayama H, et al. Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol. 2000, 35:840～848
6. Potapenko IO, Haakensen VD, Luders T, et al. Glycan gene expression signatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 2010, 4:98～118
7. Galfre G and Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol.1981,73:3
8. Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005, 41:207～234
1. Springer G F. T and Tn, general carcinoma autoantigens. Science. 1984, 224(4654):1198～206
2. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochimica Biophysica Acta. 1999. 1473(1):67～95
3. Hollingsworth M A, Swanson B J. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer. 2004, 4(1): 45～60
4. Brockhausen I. Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO reports. 2006, 7(6):599～604
5. Hector F Valenzuela, Karen E Pace, Paula V Cabrera, et al. O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1. Cancer Res. 2007, 67: (13):6155～6162
6. E Rajpert-De Meyts, S N Poll, I Goukasian, et al. Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation. Virchows Arch. 2007, 451:805～814
7. Liying Chen, Jonas Sundback, Sigvard Olofsson, et al. Interference with O-glycosylation in RMA lymphoma cells leads to a reduced in vivo growth of the tumor. Int J Cancer. 2006, 119:1495～1500
8. Georgios Patsos, Virginie Hebbe-Viton, Catherine Robbe-Masselot, et al. O-Glycan inhibitors generate aryl-glycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines. Glycobiology. 2009,19(4): 382～398
9. Hanisch F G, Reis C A, Clausen H, et al. Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides. Glycobiology. 2001, 11(9):731～740
10. Takuhiro Kohsaki, Isao Nishimori, Hirofumi Nakayama, et al. Expression of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol. 2000, 35:840～848
11. Potapenko I O, Haakensen V D, Ludersc T, et al. Glycan gene expressionsignatures in normal and malignant breast tissue; possible role in diagnosis and progression. Mol Oncol. 2010, 4(2): 98～118
12. Qiu H, Guo X H, Mo J H, et al. Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation. Glycoconj J. 2006, 23(7-8):575～584
13. Schneider U, Schwenk H, Bornkamm G. Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer. 1977, 19 (5): 621～626
14. Yamada K, Kinoshita M, Hayakawa T, et al. Comparative Studies on the Structural Features of O-Glycans between Leukemia and Epithelial Cell Lines. Journal of Proteome Research. 2009, 8(2):521～537
15. Hannon G J. RNA interference. Nature. 2002, 418(6894):244～251
16. Naldini L, Bl?mer U, Gallay P, et al. In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector. Science. 1996, 272(5259): 263～267
17. Herold M J, van den Brandt J, Seibler J, et al. Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc Natl Acad Sci USA. 2008, 105(47): 18507～18512
18. Hitomi Tsuiji, Seiichi Takasaki, Michiie Sakamoto, et al. Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinoma-associated antigen, and facilitates cell-cell adhesion. Glycobiology. 2003, 13(7):521～527
19. Phillips ML, Nudelman E, Gaeta FCA, et al. ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex. Science. 1990, 250: 1130～1132
20. Takada A, Ohmori K, Takahashi N, et al. Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A. Biochem Biophys Res Commun. 1991; 179: 713～719
21. Takada A, Ohmori K, Yoneda T, et al. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res. 1993; 53: 354～361
22. Kannagi R. Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer. Glycoconj J. 1997; 14: 577～584
23. Ito K, Ye CL, Hibi K, Mitsuoka C, et al. Paired tumor marker of soluble E-selectin and its ligand sialyl Lewis A in colorectal cancer. J Gastroenterol. 2001; 36: 823～829
24. Reiji Kannagi, Mineko Izawa, Tetsufumi Koike, et al. Carbohydrate-mediated cell adhesion in cancer metastasis and angiogenesis. Cancer Sci. 2004,95(5):377～384
1. Turnbull J E, Field A R. Emerging glycomics technologies. Nature chemical biology, 2007, 3(2): 74～77
2. Ohtsubo K, Marth J D. Glycosylation in cellular mechanisms of health and disease. Cell, 2006, 126(5): 855～867
3. Marth J D, Grewal P K. Mammalian glycosylation in immunity. Nat Rev Immunol, 2008, 8(11): 874-87
4. Haltiwanger R S, Lowe J B. Role of glycosylation in development. Annu Rev Biochem, 2004, 73: 491～537
5. Wopereis S, Lefeber D J, Morava E, et al. Mechanisms in protein O-glycan biosynthesis and clinical and molecular aspects of protein O-glycan biosynthesis defects: A Review. Clinical Chemistry, 2006, 52(4): 574～600
6. Hollingsworth M A, Swanson B J. Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer, 2004, 4(1): 45～60
7. Ju T, Brewer K, D’Souza A, et al. Cloning and expression of human core 1β1,3-galactosyltransferase. J Biol Chem, 2002, 277(1): 178～186
8. Narimatsu Y, Ikehara Y, Iwasaki H, et al. Immunocytochemical analysis for intracellular dynamics of C1GalT associated with molecular chaperone, Cosmc. Biochem Biophys Res Commun, 2008, 366(1): 199～205
9. Ju T, Cummings R D. Protein glycosylation: chaperone mutation in Tn syndrome. Nature, 2005, 437(7063): 1252
10. Schietinger A, Philip M, Yoshida B A, et al. A mutant chaperone converts a wild-type protein into a tumor-specific antigen. Science, 2006, 314(5797): 304～308
11. Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells. Biochim Biophys Acta, 1999, 1473(1): 67～95
12. Fukuda M. Roles of mucin-type O-glycans synthesized by core2beta1,6-N-acetylglucosaminyltransferase. Methods Enzymol, 2006, 416: 332～346
13. Ten Hagen K G, Fritz T A, Tabak L A. All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology,2003, 13(1): 1R～16R
14. Homa F L, Hollander T, Lehman D J, et al. Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminy- ltransferase. J Biol Chem, 1993, 268(17): 12609～12616
15. Hagen F K, VanWuyckhuyse B, Tabak L A. Purification, cloning and expression of a bovine UDP-GalNAc:polypeptide N-Acetylgalactosaminyltransferase. J Biol Chem, 1993, 268(25): 18960～18965
16. Hagen F K, Gregoire C A, Tabak L A. Cloning and sequence homology of a rat UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. Glycoconj J, 1995, 12(6): 901～909
17. White T, Bennett E P, Takio K, et al. Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem, 1995, 270(41): 24156～24165
18. Hagen F K, Ten Hagen K G, Beres T M, et al. cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminy- ltransferase. J Biol Chem, 1997, 272(21): 13843～13848
19. Yoshida A, Hara T, Ikenaga H, et al. Cloning and expression of a porcine UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase. Glycoconj J, 1995, 12(6): 824～828
20. Bennett E P, Hassan H, Clausen H. cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3. J Biol Chem, 1996, 271(29): 17006～17012
21. Zara J, Hagen F K, Ten Hagen K G, et al. Cloning and expression of mouse UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase-T3. Biochem Biophys Res Comm, 1996, 228(1): 38～44
22. Bennett E P, Hassan H, Mandel U, et al. Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine: polypeptide Nacetylgalactosaminyltransferase that complements other GalNActransferases in complete O-glycosylation of the MUC1 tandem repeat. J Biol Chem, 1998, 273(46): 30472～30481
23. Ten Hagen K G, Hagen F K, Balys M M, et al. Cloning and expression of a novel, tissue specifically expressed member of the UDP-GalNAc: polypeptide N-acetylgala ctosaminyltransferase family. J Biol Chem, 1998, 273(42): 27749～27754
24. Bennett E P, Hassan H, Mandel U, et al. Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine: polypeptide Nacetylgalactosaminyltransferase-T3, designated GalNAc-T6. J Biol Chem, 1999, 274(36): 25362～25370
25. Ten Hagen K G, Tetaert D, Hagen F K, et al. Characterization of a UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases that displays glycopeptide N-acetylgalactosaminyl transferases activity. J Biol Chem, 1999, 274(39): 27867～27874
26. Bennett E P, Hassan H, Hollingsworth M A, et al. A novel human UDP-N-acetyl-D-galactosamine:polypeptide Nacetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates. FEBS Letters, 1999, 460(2): 226～230
27. White K E, Lorenz B, Evans W E, et al. Molecular cloning of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T8, and analysis as a candidate autosomal dominant hypophosphatemic rickets (ADHR) gene. Gene, 2000, 246(1-2): 347～356
28. Toba S, Tenno M, Konishi M, et al. Brain-specific expression of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9). Biochim Biophys Acta, 2000, 1493(1-2): 264～268
29. Zhang Y, Iwasaki H, Wang H, et al. Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminy- ltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen. J Biol Chem, 2003, 278(1): 573～584
30. Ten Hagen K G., Bedi G S, Tetaert D, et al. Cloning and characterization of a ninth member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases family, ppGaNTase-T9. J Biol Chem, 2001, 276(20):17395～17404
31. Cheng L M, Tachibana K, Zhang Y, et al. Characterization of a novel humanUDP-GalNAc transferase, pp-GalNAc-T10. FEBS Lett, 2002, 531(2): 115～121
32. Schwientek T, Bennett E P, Flores C, et al. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminy- ltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila. J Biol Chem, 2002, 277(25): 22623～22638
33. Guo J M, Zhang Y, Cheng L, et al. Molecular cloning and characterization of a novel member of the UDP-GalNAc: Polypeptide N-acetylgalactosaminy- ltransferase family, pp-GalNAc-T12. FEBS Letters, 2002, 524(1-3): 211～218
34. Hennet T, Hagen F K, Tabak L A, et al. T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination. Proc Natl Acad Sci USA, 1995, 92(26): 12070～12074
35. Wang H, Tachibana K, Zhang Y, et al. Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase,pp-GalNAc-T14. Biochem Biophys Res Commun, 2003, 300(3): 738～744
36. Cheng L, Tachibana K, Iwasaki H, et al. Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15. FEBS Lett, 2004, 566(1-3): 17～24
37. Wang J, Shao D, Xie C, et al. The research of a novel UDP-N-acetyl-α-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase like gene. 21st IUBMB and 12st FAOBMB International Congress of Biochemistry and Molecular Biology, Shanghai, August 2-7, 2009[C]. pp195: E-2-020
38. Peng C, Togayachi A, Kwon Y D, et al. Cloning and characterization of a novel human UDP-GalNAc transferase, ppGalNAc-T20. 21st IUBMB and 12st FAOBMB International Congress of Biochemistry and Molecular Biology, Shanghai, August 2-7, 2009[C]. pp195: E-2-022
39. R(?)ttger S, White J, Wandall H H, et al. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus. J Cell Sci, 1998, 111(Pt1): 45～60
40. Zhou Y H, Hang S Y, Qiu H, et al. UDP-GalNAc: polypeptideα-N-acetygalactosaminyltransferase 2 localized on both cis and trans side of Golgi stacks in SGC7901 cells. Progress in Biochemistry and Biophysics, 2009, 36(1): 49～57
41. Wandall H H, Irazoqui F, Tarp M A, et al. The lectin domains of polypeptide GalNAc-transferases exhibit carbohydrate-binding specificity for GalNAc: Lectin binding to GalNAc-glycopeptide substrates is required for high density GalNAc-O-glycosylation. Glycobiology, 2007, 17(4): 374～387
42. Fritz T A, Hurley J H, Trinh L B, et al. The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminy- ltransferase-T1. Proc Natl Acad Sci USA, 2004, 101(43), 15307～15312
43. Fritz T A, Raman J, Tabak L A. Dynamic association between the catalytic and lectin domains of human UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminy- ltransferase-2. J Biol Chem, 2006, 281(13): 8613～8619
44. Kubota T, Shiba T, Sugioka S, et al. Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminy- ltransferase (pp-GalNAc-T10). J Mol Biol, 2006, 359(3): 708～727
45. Marchler-Bauer A, Bryant S H. CD-Search: protein domain annotations on the fly. Nucleic Acids Res, 2004, 32: W327～331
46. Berois N, Mazal D, Ubillos L, et al. UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker. J Histochem Cytochem, 2006, 54(3): 317～328
47. Inoue T, Eguchi T, Oda Y, et al. Expression of GalNAc-T3 and its relationships with clinicopathological factors in 61 extrahepatic bile duct carcinomas analyzed using stepwise sections - special reference to its association with lymph node metastases-. Mod Pathol, 2007, 20(2): 267～276
48. Gomes J, Marcos N T, Berois N, et al. Expression of UDP-N-acetyl-D-galactosamine: Polypeptide N-acetylgalactosaminyltransferase-6 in gastric mucosa, intestinal metaplasia, and gastric carcinoma, Journal of Histochem Cytochem, 2009, 57(1): 79～86
49. Guo J M, Chen H L, Wang G M, et al. Expression of UDP-GalNAc: Polypeptide N-Acetylgalactosaminyl transferase-12 in gastric and colonic cancer cell lines and in human colorectal cancer. Oncology, 2004, 67(3-4): 271～276
50. Qiu H, Guo X H, Mo J H , et al. Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during1,25-dihydroxyvitamin D3 induced differentiation. Glycoconj J, 2006, 23(7-8): 575～584
51. Shimodaira K, Nakayama J, Nakamura N, et al. Carcinoma-associated expression of core 2β-1,6-Nacetylglucosaminyltransferase gene in human colorectal cancer: role of O-glycans in tumor progression. Cancer Res, 1997, 57(23): 5201～5206
52. Brockhausen I. Mucin-type O-glycans in human colon and breast cancer: glycodynamics and functions. EMBO reports, 2006,7(6): 599-604
53. Wu L H, Hu P, Wu W, et al. Fucosylated glycans associated with development and metastasis of hepatocellular carcinoma cells. Progress in Biochemistry and Biophysics, 2009, 36(1): 33～41
54. Kanoh A, Takeuchi H, Kato K, et al. Interleukin-4 induces specific pp-GalNAc-T expression and alterations in mucin O-glycosylation in colonic epithelial cells. Biochimica et Biophysica Acta, 2008, 1780(3): 577～584
55. Higai K, Miyazaki N, Azuma Y, et al. Interleukin-1βinduces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene. FEBS Letters, 2006, 580(26): 6069～6075
56. Wu Y M, Nowack D D, Omenn G S, et al. Mucin glycosylation is altered by pro-inflammatory signaling in pancreatic-cancer cells. Journal of Proteome Research, 2009, 8(4):1876～1886
57. van Kooyk Y, Rabinovich G A. Protein-glycan interactions in the control of innate and adaptive immune responses. Nature Immunology, 2008, 9(6): 593-601
58. Tsuji H, Takasaki S, Sakamoto M, et al. Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinomaassociated antigen, and facilitates cell-cell adhesion. Glycobiology, 2003, 13(7): 521～527
59. Bao X F, Kobayashi M, Hatakeyama S, et al. Tumor suppressor function of laminin-bindingα-dystroglycan requires a distinctβ3-Nacetylglucosaminy- ltransferase. Proc Natl Acad Sci USA, 2009, 106(29):12109～12114
60.朱江龙, Danishefsky S J.前列腺癌和乳腺癌的免疫治疗新进展.科学通报, 2008, 53 (17): 2126