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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林俊宏(Chun-Hung Lin) | |
dc.contributor.author | Chia-Ling Liu | en |
dc.contributor.author | 劉家玲 | zh_TW |
dc.date.accessioned | 2021-06-13T03:28:56Z | - |
dc.date.available | 2006-07-31 | |
dc.date.copyright | 2006-07-31 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-27 | |
dc.identifier.citation | 第五章 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32036 | - |
dc.description.abstract | 幽門螺旋桿菌 (Helicobacter pylori,簡稱H. pylori)感染世界上50%的人口,目前被證實感染幽門桿菌會造成消化性潰瘍、胃淋巴瘤等相關疾病。這株致病菌可以在LPS (lipopolysaccharide) 結構末端的O抗原上表現路易士抗原 (Lewis antigen),這種路易士抗原可以在感染週遭的胃上皮細胞看到,如此一來,幽門桿菌就可以藉模擬而躲過宿主細胞的免疫系統攻擊。在路易士抗原合成途徑中,岩藻醣轉移酶負責催化最後幾步的反應,可以將GDP-Fuc的fucose轉移到受質上。α-1,3-fucosyltransferase (簡稱α-1,3-FucT)以LacNAc為受質,經轉糖作用後形成Lex。α-1,3-FucT在C端有兩性螺旋序列,可以幫助蛋白質附著於膜上。本實驗室之前將蛋白質從C端做45個胺基酸的截短,得到水溶性蛋白質。此種長度的截短我們稱之為C45-FucT,與具有全長的α-1,3-FucT得到具有相同的高活性。從兩性螺旋序列往N端找,可以看到十組七個胺基酸的重複序列。C115-FucT從C端截短115個胺基酸,包含兩性螺旋序列與可以形成Leu zipper的組七個胺基酸的重複序列;由於C115-FT失去可以幫助形成雙體的Leucine zipper,故此種長度的截短酵素為單體且活性下降到6.5 %。
由於缺乏FucT的結構資訊,所以要了解此酵素的活性區與催化機制,是件不容易的事。利用化學修飾法找出His和Arg可能參與受質的結合後,我們將10個His與10個Arg分別利用定點突變並做活性測試,討論與GDP-Fuc和LacNAc結合有關的胺基酸。 DEPC (diethylpyrocarbonate) 和PGO (phenylglycoxal) 分別會與His和Arg形成共價鍵進而抑制酵素反應。但是當α-1,3-FucT先與GDP-Fuc結合達到保護donor sugar結合區後,再加入DEPC或PGO,抑制的效果卻會大大減低。由於GDP-Fuc的保護作用,我們可以推測在GDP-Fuc結合區,有某些His和Arg幫助GDP-Fuc跟蛋白質結合。 我們將C45-FucT的10個Arg與10個His突變成Ala並分別測試活性。活性測試是利用FucT的副產物GDP的生成量會造成NADH的減少,藉由測量NADH的量而達到測試FucT活性的目的。其中R79A、 R118A、R180A、R195A 和R354A的突變株活性完全消失,這五個胺基酸對α-1,3-FucT的活性是非常重要的。R89A、H60A、H131A、H282A、H298A和H345A則是有活性下降。我們將這六個活性下降的突變株更進一步測Km。由Km的數據得知H282、H298和H354會幫助GDP-Fuc結合;H60與R89會幫助LacNAc結合。 最近,王惠鈞教授實驗室將FucT做出結晶解出結構。目前為止只有C115-FucT這種截短的酵素可以得到結晶。從結構上,我們看到R195、N240、E249和K250會直接跟GDP-Fuc有氫鍵結合。但是在活性測試的實驗中卻發現N240會幫助LacNAc與酵素作用。利用結構疊合,我們推斷E95為catalytic base。另外藉由酵素動力學測試和C115-FucT的結構,我們推測出更接近野生型α-1,3-FucT的反應機制與構型。 | zh_TW |
dc.description.abstract | Helicobacter pylori (H. pylori) is considered to be a primary cause of gastritis, duodenal ulcer and gastric cancer. This pathogenic bacterium produces Lex and Ley epitopes in the O-antigens of lipopolysaccharides (LPS) to mimic the carbohydrate antigens on the surface of gastric epithelial cells. The molecular mimicry avoids the detection by the host immune system. The enzyme α-1,3-fucosyltransferase (α1,3-FucT) from H. pylori catalyzes the glycosyl addition of fucose from the donor GDP-fucose to the acceptor LacNAc. It is a membrane associated protein. Previously, our lab carried out a systematic truncation of the C terminus of the α-1,3-FucT to improve the protein solubility. C-Terminal 45 amino acid residues include the amphiphilic helices that anchor the protein to the cell membrane. The corresponding protein, C45-FucT, was water soluble and had the same activity as that of the full-length enzyme. Next to the amphiphilic helices is the heptad repeats. Deletion of 115 residues removed both helices and the heptad repeats in the C terminal end. Because heptad repeats were expected to form the leucine zipper and facilitated the formaitnon of a dimeric structure, therefore C115-FucT was found to be a monomer with low activity.
Due to lack of structural imformation of fucosyltransferase, it is not easy to understand the catalytic mechanism. In order to study the residues involed in catalysis and substrate binding, this thesis lays a special emphasis on His and Arg. The methods include the use of modifiers and preparation of different mutant proteins by site-directed mutagenesis. DEPC (diethylpyrocarbonate) and PGO (phenylglycoxal) were shown to specifically react with His and Arg, respectively. Both of these modifier can inactivate the α-1,3-FucT of H. pylori. When α-1,3-FucT was incubated with GDP-Fuc before the addition of PGO or DEPC, the higher concentration of GDP-Fuc was used in the study, the higher level of activity was retained. The protection effect of GDP-Fuc indicated that His and Arg were likely located at either the active site or the binding site of the donor substrate. Ten Arg residues and ten His residues were individually mutated to Ala and the resulting mutants were analyzed by the activity assay. The activity assay was a coupling assay carried out based on the production of the side product GDP. The result indicated the mutants R79A, R118A, R180A, R195A and R354A were completely inactive. R89A, H60A, H131A, H282A, H298A and H345A were found to have reduced activities. The Km and Vmax of these mutants were also measured. According to Km, H282, H298 and H354 were found to be involved in the binding with GDP-Fuc, Whereas H60 and R89 were related to the binding with LacNAc. The structure of was solved by x-ray crystallograpgy in the collaboration with Dr. Andrew Wang’s group of this institute. So far only C115-FucT can be crystallized for prove the structural imformation. The results suggest that R195, N240, E249, K250 directly interact with GDP-Fuc. In contrast, the kinetic analysis supports the idea that N240 is involved in the binding with LacNAc. The superimposition of C115-FucT to the structure of BGT (β-Glucosyltransferase) revealed that E95 may act as the catalytic base in general catalytic base. We also try to figure out the real structure of the C45-FucT. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:28:56Z (GMT). No. of bitstreams: 1 ntu-95-R92b46026-1.pdf: 5807390 bytes, checksum: 37dd0c982cbd3c459cee46304d8fefc6 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 目 錄
目錄 I 縮寫表 1 中文摘要 3 英文摘要 5 第一章 序論 7 1. 幽門螺旋桿菌 7 1. 幽門螺旋桿菌之簡介 7 2. 幽門螺旋桿菌之感染 7 2. 路易士抗原 8 1. 血型抗原與路易士抗原 8 2. 血型抗原與癌症 9 3. 幽門螺旋桿菌與路易士抗原 9 3. 岩藻醣轉移酶 10 1. 岩藻醣轉移酶的簡介 10 2. 人類α-1,2-FucT與α-1,6-FucT 11 3. 人類α-1,3-FucT、α-1,4-FucT與α-1,3/4-FucT 11 4. 幽門螺旋桿菌的α-1,2-FucT 12 5. 幽門螺旋桿菌的α-1,3-FucT、α-1,4-FucT、α-1,3/4-FucT 12 4. 醣轉移酶與其結構 14 1. 醣轉移酶與分類法 14 2. 醣轉移酶的二極與三極結構 15 3. GT-A superfamily 與CstII 15 4. GT-B superfamily 16 5. 實驗動機與目的 19 第二章 材料與方法 21 1. 實驗材料 21 2. 實驗之儀器設備 21 3. 分子生物學實驗 22 4. 定點突變基因的建構 23 5. 蛋白質之表現 25 6. 蛋白質之純化 25 7. 蛋白質之鑑定 27 8. 酵素活性與動力學之分析 28 第三章 實驗結果 31 1. 二級結構比對與以結構決定重要之胺基酸與設計突變用引子 31 2. 定點突變株之建構結果 31 3. 蛋白質表現與純化之結果 32 4. His與Arg突變蛋白質活性分析之結果 32 5. 與GDP-Fuc結合之重要胺基酸的突變蛋白質活性分析之結果 33 6. Km與Vmax的酵素動力學測試 33 7. Catalytic base的酵素動力學研究 34 第四章 結果與討論 35 1. 利用化學修飾劑作用與胺基酸突變來研究岩藻醣轉移酶 35 2. 蛋白質結構 37 3. catalytic base與α-1,3-FucT的催化機制 40 4. 整體活性區與蛋白質結構之探討 40 第五章 參考文獻 87 圖次 圖一、A、B、H determinant之結構 43 圖二、第一型與第二型之路易士抗原結構 44 圖三、真核生物與原核生物岩藻醣轉移酶之比較 45 圖四、α-1,2-FucT與α-1,6-FucT之相似序列 46 圖五、α-1,3-FucT之相似序列 47 圖六、推測FucT-V的催化機制 48 圖七、人類與幽門螺旋桿菌合成路易士抗原之途徑 49 圖八、LPS之結構 50 圖九、α-1,3-FucT不同的截短形式與結構卡通圖 51 圖十、幽門螺旋桿菌α-1,3-FucT與α-1,3/4-FucT之胺基酸序列比較 52 圖十一、Rossmann-type fold 53 圖十二、醣轉移酶之結構 54 圖十三、GT-A的催化機制 55 圖十四、BGT的蛋白質結構 56 圖十五、定點突變基因的建構 57 圖十六、BGT與C115-FucT的superimpose 58 圖十七、C115-FucT的結構 59 圖十八、以NADH為基礎並偶合丙酮酸激酶/乳酸脫氫酶的螢光測試 60 圖十九、GDP-Fuc的結合胺基酸 61 圖二十、PGO與C45-FucT作用 62 圖二十一、DEPC與C45-FucT作用 63 圖二十二、比較BGT與C115-FucT之兩個Rossmann-type fold結構 64 圖二十三、motif在人類 FucT-VI與H. pylori C45-FucT之突變株活性比較 65 圖二十四、H. pylori α-1,3-FucT的催化機制 66 圖二十五、與GDP-Fuc結合有關的胺基酸 67 圖二十六、與LacNAc結合有關的胺基酸 68 圖二十七、推測C45-FucT的結構修正 69 表次 表一、幽門螺旋桿菌與其表現之路易士抗原 71 表二、人類岩藻糖轉移酶 72 表三、目前已發表之醣轉移酶結構 73 表四、引子序列表 74 表五、GT結構的分類與預測 76 表六、與GDP-Fuc有氫鍵之胺基酸 77 表七、Arg與His突變株活性測試百分比 78 表八、和GDP-Fuc有氫鍵結合的突變株與catalytic base的活性測試百分比 79 表九、與GDP-Fuc的Km有改變的突變株 80 表十、與LacNac的Km有改變的突變株 81 附錄 附錄一、NCTC 11639 Helicobacter pylori α1,3-FT之基因序列 83 附錄二、NCTC 11639 Helicobacter pylori α1,3-FT之基因序列 84 | |
dc.language.iso | zh-TW | |
dc.title | 利用定點突變研究幽門螺旋桿菌的岩藻糖轉移酶之活性區 | zh_TW |
dc.title | Study the active site of Helicobactor pylori
α-1,3-fucosyltransferase by site-directed mutagenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 邱式鴻,邱繼輝,陳玉如 | |
dc.subject.keyword | 幽門螺旋桿菌,岩藻榶轉移酶, | zh_TW |
dc.subject.keyword | Helicobacter pylori,fucosyltransferase, | en |
dc.relation.page | 92 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-28 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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