林俊宏Lin, Chun-Hung臺灣大學:生化科學研究所劉大偉Liu, Ta-WeiTa-WeiLiu2010-05-042018-07-062010-05-042018-07-062009U0001-0308200917493800http://ntur.lib.ntu.edu.tw//handle/246246/178862全球約有50%的人口感染幽門螺旋桿菌；研究顯示，這個病原菌與胃炎、十二指腸潰瘍與胃癌的形成，有直接的相關性。幽門螺旋桿菌與宿主細胞間如何傳達訊息而互相影響？再者，這種交互作用的機制，是否對於不同胃部疾病的形成有關？這些問題到目前為止還不是很清楚。利用對含有岩藻醣的複合醣做螢光標示，我們發現幽門螺旋桿菌可以從宿主細胞攝入岩藻醣，在共同培養幽門螺旋桿菌與宿主細胞的條件下（以幽門螺旋桿菌去感染宿主細胞），宿主細胞會分泌出第二型岩藻醣水解酶到共同培養液中。利用RNA干擾技術降低宿主細胞第二型岩藻醣水解酶的表現，可以降低CagA（幽門螺旋桿菌的一種毒性分子）轉移到宿主細胞內的比例。因此，第二型岩藻醣水解酶的存在可能與幽門螺旋桿菌附著到宿主細胞表面的能力有關，尤其對從十二指腸潰瘍與胃癌病人取出的幽門螺旋桿菌株有所影響。另外，第二型岩藻醣水解酶的表現，可以增加幽門螺旋桿菌路易士X抗原的形成，此類醣分子除了加強對宿主細胞的附著能力，還可以作為偽裝胃壁細胞表面的醣分子種類，藉此躲避宿主免疫系統的攻擊。這些發現，不只證實了宿主第二型岩藻醣水解酶對於幽門螺旋桿菌的附著、生長與致病的能力有關，也說明了此酵素對於幽門螺旋桿菌所導致的相關疾病，可以作為一個臨床診斷與治療潛力的標的物。Infecting about one-half of the global human population, Helicobacter pylori is well established as the primary cause of gastritis, duodenal ulcer and gastric cancer. Currently there is no clear information regarding if and how host cells interact with H. pylori and if such interactions are dependent on the type of gastric disease. Using fluorescently labeled fucose-containing glycoconjugates, we provide the first evidence observing both the uptake of L-fucose from gastric cancer cells to H. pylori and that human α-L-fucosidase 2 (FUCA2) is secreted only under co-culture conditions (i.e., host cells infected with H. pylori). Upon depletion of FUCA2 by RNA interference and detection of translocated CagA (a virulence factor of H. pylori) in host cells, FUCA2 was found to be essential for H. pylori adhesion, in particular to the gastric cancer- and duodenal ulcer-specific strains. Additionally FUCA2 was shown to significantly enhance the expression of Lewis x antigen in H. pylori, which is critical for the bacterial cell adhesion in the pathogenesis and defense strategy to escape from host surveillance. These findings not only demonstrate an important connection between FUCA2 and the adhesion, growth and pathogenicity of H. pylori, but also support the idea that FUCA2 is a potential target for clinical diagnosis and therapeutic intervention of H. pylori-related diseases.中文摘要 (1)bstract (2). Introduction.1. Role of Helicobacter pylori in the pathogenesis of gastritis, peptic ulcer and gastric cancer (11).2. Molecular basis for host recognition and bacterial adherence of H. pylori (11).3. H. pylori Lewis antigens as adhesions (14).4. Lewis antigens and putative autoimmune mechanisms (19).5. Lewis antigens mimicry and immune evasion: the example of Bacteroides thetaiotaomicron (21).6. Specific finding (24). Results.1. H. pylori extracts 6-azido-L-fucose from human epithelial fucosylated glycoconjugates (26).2. α-L-Fucosidase is secreted from host cells in response to H. pylori infection (27).3. Secreted α-L-fucosidase controls H. pylori adhesion (29).4. α-L-Fucosidase activity is critical for H. pylori attachment in strains isolated from patients with duodenal ulcers or gastric cancer (30).5. α-L-Fucosidase activity regulates the expression level of Lex antigen (32). Discussion.1. Role for human α-L-fucosidase 2 (FUCA2) in the control of H. pylori-infected gastric cancer cells (34).2. A role foe host glycans in commensal relationships (36).3. Antibiotic treatment of H. pylori infection: the potential of α-L-fucosidase inhibitor (38). Materials and methods.1. Bacterial strains and cell lines (39).2. H. pylori and cancer cell co-culture conditions (39).3. Confocal fluorescence microscopy and imaging (40).4. Purification and activity assay of α-L-fucosidase (41).5. In-gel digestion (42).6. Mass spectrometry analysis (44).7. Cloning, expression and purification of human FUCA2 (45).8. Kinetic analysis of human FUCA1 and FUCA2 (46).9. RNA interference (47).10. Flow cytometric analysis of fucosylated glycans on the surface of H. pylori or Capan 1 cells (48).11. Infection assays and immunoblotting of CagA and Lex(49). References (52)able 1 Ki values of L-fucose, 6-azido-L-fucose and 1-aminomethyl-1-deoxy-fuconojirimycin (FNJ) for human FUCA1 and FUCA2 (69)ig. 1A Schematic representation showing metabolic incorporation of tetra-O-acetyl-6-azido-L-fucose (70)ig. 1B Procedure for specific fluorescent labeling of fucosylated glycans in cells (70)ig. 2 Fluorescent imaging of fucosylation by incubating AGS, N87 and Capan 1 cells with tetra-O-acetyl-6-azido-L-fucose (72)ig. 3 H. pylori obtains L-fucose from host cell membranes (4h) (73)ig. 4 H. pylori takes up L-fucose from the host cell (8h) (75)ig. 5 H. pylori takes up 6-azido-L-fucose directly from the host culture medium (76)ig. 6A Activity assay of the secreted α-L-fucosidase from Capan 1 cells co-cultured with H. pylori (78)ig. 6B Activity assay of the secreted α-L-fucosidase from five gastric adenocarcinoma cell lines cells uninfected with H. pylori (79)ig. 6C Activity assay of the secreted α-L-fucosidase from five gastric adenocarcinoma cell lines cells co-cultured with H. pylori (80)ig. 6D The enzymatically active pooled fractions obtained from the affinity chromatography-based purification were analyzed by SDS-PAGE (80)ig. 6E Identification of α-L-fucosidase 2 (FUCA2) (81)ig. 7 Sequence alignment of human α-L-fucosidase 1 (FUCA1) and α-L-fucosidase 2 (FUCA2) (83)ig. 8A SDS-PAGE analysis of purified human FUCA1 and FUCA2 (84)ig. 8B Double reciprocal plot of L-fucose for the Ki value of FUCA1 (84)ig. 8C Double reciprocal plot of 6-azido-L-fucose for the Ki value of FUCA1 (85)ig. 8D Double reciprocal plot of L-fucose for the Ki value of FUCA2 (85)ig. 8E Double reciprocal plot of 6-azido-L-fucose for the Ki value of FUCA2 (86)ig. 8F Double reciprocal plot of FNJ for the Ki value of FUCA2 (86)ig. 9 Secreted FUCA2 is essential for H. pylori adhesion to host cells (4h) (88)ig. 10A FUCA2 is essential for H. pylori adhesion to host cells (8h) (90)ig. 10B Immunoblot analysis of mock-transfected Capan 1 cells infected with H. pylori using mouse monoclonal anti-CagA (91)ig. 10C Immunoblot analysis of Capan 1-FUCA2 K.D. cells infected with H. pylori using mouse monoclonal anti-CagA (91)ig. 11A Treatment of Capan 1 cells with FNJ was found to considerably reduce the transfer of 6-azido-L-fucose from host cells to H. pylori (93)ig. 11B Flow cytometric analysis of fucosylated glycoconjugates on the surface of H. pylori (94)ig. 11C Flow cytometric analysis of fucosylated glycoconjugates on the surface of Capan 1 cells (94)ig. 12A Immunoblot analysis of Capan 1 cells infected with different gastric syndrome H. pylori strains using mouse monoclonal anti-CagA (96)ig. 12B Immunoblot analysis of Capan 1 cells infected with different gastric syndrome H. pylori strains using mouse monoclonal anti-CagA (with 100 μM FNJ) (96)ig. 12C Immunoblot analysis of AGS cells infected with different gastric syndrome H. pylori strains using mouse monoclonal anti-CagA (97)ig. 12D Immunoblot analysis of AGS cells infected with different gastric syndrome H. pylori strains using mouse monoclonal anti-CagA (with 100 μM FNJ) (97)ig. 13A Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains-infected Capan 1 cells with mouse monoclonal anti-CagA (99)ig. 13B Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains-infected Capan 1 cells with mouse monoclonal anti-CagA (with 100 μM FNJ) (99)ig. 13C Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains-infected AGS cells with mouse monoclonal anti-CagA (100)ig. 13D Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains-infected AGS cells with mouse monoclonal anti-CagA (with 100 μM FNJ) (100)ig. 14A Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains -infected mock-transfected Capan 1 cells with mouse monoclonal anti-Lex antigen (102)ig. 14B Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains -infected Capan 1-FUCA2 K.D. cells with mouse monoclonal anti-Lex antigen (102)ig. 15A Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains -infected Capan 1 cells with mouse monoclonal anti-Lex antigen (104)ig. 15B Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains -infected Capan 1 cells with mouse monoclonal anti-Lex antigen (with 100 μM FNJ) (104)ig. 15C Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains-infected AGS cells with mouse monoclonal anti-Lex antigen (105)ig. 15D Immunoblot analysis of different H. pylori duodenal ulcer (DU) strains-infected AGS cells with mouse monoclonal anti-Lex antigen (with 100 μM FNJ) (105)ig. 16 Immunoblot analysis of Capan 1 cells infected with H. pylori and blotted with anti-Fucosyltransferase 3/6 antibody (107)application/pdf5579839 bytesapplication/pdfen-US幽門螺旋桿菌岩藻醣岩藻醣水解酶毒性分子胃部疾病Helicobacter pylorifucosefucosidasevirulencegastric disease[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178862/1/ntu-98-D93b46005-1.pdf