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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56817完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊宏 | |
| dc.contributor.author | Cheng-Guo Wu | en |
| dc.contributor.author | 吳正國 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:50:22Z | - |
| dc.date.available | 2019-09-04 | |
| dc.date.copyright | 2014-09-04 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-08 | |
| dc.identifier.citation | 1 Delves, P. J. The role of glycosylation in autoimmune disease. Autoimmunity
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56817 | - |
| dc.description.abstract | 岩藻醣為經常修飾在寡糖鏈末端的結構，並賦予與之鍵結寡醣鏈特定的生化活性，因為岩藻醣基化之醣鏈在許多生理作用上扮演不可或缺的重要性，因此與岩藻醣基化寡糖鏈生合成相關之酵素對生物非常的重要。岩藻醣基化作用由三步驟反應完成：第一，岩藻醣激酶磷酸化岩藻醣形成岩藻醣-1-磷酸；第二，鳥苷二磷酸岩藻醣焦磷酸化酶催化岩藻醣-1磷酸成鳥苷二磷酸岩藻糖；第三，岩藻糖轉移酶將岩藻醣轉移至醣鏈受質，完成反應。相反的，岩藻糖水解酶則負責將岩藻醣從醣鏈上水解移除。本論文著重在探討來自鬆脆桿菌 (Bacteroides fragilis) 的鳥苷二磷酸岩藻醣合成酶及人類第二型岩藻糖水解酶。此兩者在宿主及細菌間之交互作用關係中皆扮演重要的角色。
鬆脆桿菌的鳥苷二磷酸合成酶簡稱FKP，是一個擁有兩個酵素活性的雙功能蛋白，同時表現在其他物種上分別表達的岩藻醣激酶及鳥苷二磷酸岩藻醣焦磷酸化酶於一個蛋白上，得以以單一酵素完成鳥苷二磷酸岩藻醣的補救合成途徑。在本研究中我們成功分別表達具有單一酵素活性的FKP區段，藉比較單一區段與全長FKP的酵素動力學參數，我們發現共同表達兩個區段於一個蛋白時的催化優勢，得以幫助細菌在競爭的腸道環境具有競爭性。此外，本研究中我們藉者解出岩藻醣激酶與岩藻醣及岩藻醣-1-磷酸的複合體結構得知，岩藻醣C-5旁有一個很大的洞穴，合理解釋以往研究發現FKP對岩藻醣C-5上之取代基有廣泛的接受度之原因，藉由結構及酵素動力學研究我們進而歸納其對各式醣類的受質專一性，發現醣類之C-1、C-3及C-4的正確立體位向對於其醣類之結合極為重要，而C-5的彈性使之得以催化岩藻醣以外之醣類。綜合以上，我們同時提供 FKP 之生物優勢及合成鳥苷二磷酸醣類衍生物的化學應用性的新觀點。 人類第二型岩藻醣水解酶 (α-L-Fucosidase) 之重要性首先由本研究室提出，我們發現FUCA2只有當胃表皮細胞被胃幽門螺旋桿菌感染時才會釋出，並對病原體獲取宿主之岩藻醣有重要幫助；為了進一步探討鮮為人研究的FUCA2生理功能，以往利用許多方式表現FUCA2，但皆受限於其表現量太低或沒有活性的因素而宣告失敗。本研究成功利用畢赤酵母菌成功表現有活性之FUCA2，並提升其蛋白質產量，進一步確認畢赤酵母菌表現之FUCA2其上至少含有3個被醣基化的位置，發現FUCA2的醣基化現象對其穩定性及活性之重要性。 進一步利用質譜分析，發現FUCA1及FUCA2對於帶有兩個岩藻醣分子的Ley及Leb抗原有最佳的催化活性，而對於只帶有一個岩藻醣的Lea和Lax活性較差，甚至對於血型抗原A、B及H沒有水解活性，這樣的結果可能暗示著人類岩藻醣水解酶對於帶有多個岩藻醣分子的醣鏈有較佳的結合及催化能力。此外，藉著以FUCA1及FUCA2對直腸癌細胞及胃癌細胞株萃取之醣鏈反應後，藉由質譜得以看到不同的醣鏈圖譜產生，一方面說明FUCA1及FUCA2得以水解細胞內含有的醣鏈外，也說明FUCA1及FUCA2受質專一性的差異，說明其兩者在生物體內可能扮演不同的生理功能。 | zh_TW |
| dc.description.abstract | L-fucose frequently exists as a terminal modification of glycan structures and confers unique functional features to the attached carbohydrates. Since fucosylated glycans are essential for a variety of biological activities, the related metabolic enzymes are of physiological significance. There are three steps involved in the biosynthesis of fucosylated glycans, namely fucokinase (to catalyze the formation of L-fucose-1-phosphate), GDP-fucose pyrophosphorylase (the formation of GDP-fucose) and fucosyltransferase. In contrast, L-fucosidase is the enzyme catalyzing the hydrolytic removal of L-fucose residues. This thesis research aims at the characterizations of Bacteroides fragilis GDP-fucose synthetase and human α-L-fucosidase 2 (FUCA2) . Both proteins are known for their involvement in the bacteria/host interactions.
GDP-fucose synthetase from Bacteroides fragilis, called FKP, is a bifunctional protein that contains two separate activity domains of fucokinase (located in C-terminus) and GDP-fucose pyrophosphorylase (C-terminus), which are generally exist as separate proteins in other organisms, involved in the salvage pathway of GDP-fucose formation. In this study, we successfully prepared various forms of the two activity domains and established the corresponding activity assay to measure the kinetic parameters. The results support the idea that catalytic efficiency explains the reason why the two activity domains co-exist in one protein, providing survival advantage in the competitive mammalian intestinal ecosystem. Especially, the X-ray crystal structures of the fucokinase complexed with fucose and fucse-1-phosphate indicated that the enzyme has open space for C-5 of L-fucose, explaining the reported flexibility for C5- substituent. The hydrogen bond interactions between FKP and C-1,C-3 and C-4 hydroxyl group of sugar are strictly essential for sugar binding and catalysis. The importance of human FUCA2 was first documented in our previous findings that the enzyme is secreted only when gastric epithelium was infected by Helicobacter pylori, and that the enzyme release is critical to the uptake of L-fucose from the human host to the bacteria. To better define and characterize the functional role of FUCA2, it is essential to prepare the recombinant form of FUCA2. However, the major challenge was mainly attributed to poor expression and extremely low enzyme activity. The successful cloning and overexpression of FUCA2 in yeast P. pastroris allowed us to obtain an active recombinant protein with an improved yield (compared to the HEK 293T cell and E. coli expression), indicating that protein glycosylation contributes to the solubility and activity. The substrate specificity of FUCA1 and FUCA2 evaluated by mass analysis indicated that both enzymes have highest catalytic activity to Leb and Ley, which possess two fucose residues. In contrast, FUCA1 and FUCA2 have lower activity to Lex and Lea, and no activity to H, A, B blood type antigens which have only one fucose. The results suggested the possibility that human fucosidases have higher affinity and activity to glycans with more than one fucose attached. | en |
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| dc.description.tableofcontents | 目錄
致謝 I 常用縮寫及中英對照表 III 摘要 V Abstract VII 目錄 IX 圖、表目錄 XV Chapter 1 緒論……………………………………………………………..…….…1 1-1岩藻醣及其生物功能……..…………………………………………………....1 1-2岩藻醣基化寡醣鏈之生合成及代謝與相關酵素…….. ……………………...1 1-3 ABO血型抗原與路易士抗原之角色…………………………………...….….4 1-3-1 ABO血型抗原 (ABO blood group antigen) ……………….……..…...…4 1-3-2路易士抗原 (Lewis antigen) ……………………….………...…...………4 1-4鬆脆桿菌鳥苷二磷酸岩藻醣合成酶及其應用………………………......……6 1-4-1 補救途徑中之鳥苷二磷酸岩藻醣合成酶……………………..……...…6 1-4-2鬆脆桿菌及其與岩藻醣間之關聯 ……………………………........….…6 1-4-3 鬆脆桿菌之鳥苷二磷酸岩藻醣合成酶…………………….........………7 1-4-4 鬆脆桿菌鳥苷酸岩藻醣合成酶之生化應用…………………..….......…8 1-5 人類岩藻醣水解酶之功能與相關疾病……………………………...………10 1-5-1人類第一型岩藻醣水解酶 (FUCA1) …………………………......…….11 1-5-2人類第二型岩藻醣水解酶 (FUCA2) ………………………….....….…12 1-5-3 人類岩藻醣水解酶的催化機制…………………………….…..………13 1-5-4人類岩藻醣水解酶之醣基化現象…………………………..……...……14 1-5-5 幽門螺旋桿菌表現之路易士抗原……………………………..….……15 1-6 論文研究動機…………………………………………………………...……16 1-6-1鬆脆桿菌鳥苷二磷藻醣合成酶 (FKP) ……………………..……..……16 1-6-2 人類第二型岩藻醣水解酶 (FUCA2) …………………………...……..17 Chapter 2 結果與討論：鬆脆桿菌鳥苷二磷酸合成酶……………..…….……..18 2-1 鬆脆桿菌鳥苷二磷酸合成酶及其片段區間重組蛋白之表現與純化 …….18 2-1-1 序列比對 (Sequence aligment) …………………………...……………18 2-1-2 質體建構 (Cloning) …………………………...…………………..……19 2-1-3 蛋白質表現 (Protein expression) …………………………...…….……19 2-1-4 蛋白質純化 (protein purification) …………………………...…………20 2-2 鬆脆桿菌重組鳥苷酸岩藻醣合成酶之活性及生化特性鑑定…………...…21 2-2-1以TLC片追蹤活性……………….…………...…………………………21 2-2-2 岩藻醣磷酸酶 (FK) 之酵素動力學參數偵測……………………….....21 2-2-3鳥苷酸岩藻醣焦磷酸化酶之酵素動力學參數偵測…….………...……22 2-2-4 鳥苷酸岩藻醣合成酶與其片段之熱穩定度探討…….………...…….. 23 2-2-5 鳥苷二磷酸岩藻醣合成酶與其片段蛋白之四級結構探討……….……24 2-3 鬆脆桿菌鳥苷酸岩藻醣合成酶中岩藻醣激酶片段之蛋白質結構…...……25 2-3-1 蛋白質晶體之培養…………………………...…………………………25 2-3-2結晶學數據…………………………...………………………………..…26 2-3-3 岩藻醣激酶整體結構 (overall structure) ………………………………26 2-3-4 岩藻醣激酶片段與岩藻醣結合之複合體結構……………………...…26 2-3-5岩藻醣激酶之反應機制 (reaction mechanism) 探討………...…..27 2-3-6鳥苷酸岩藻醣合成酶對醣類之受質專一性………………………….…28 2-3-7 岩藻醣激酶片段與岩藻醣-1-磷酸結合之複合體結構…………..……30 2-3-8 岩藻醣激酶區段與其同源蛋白之序列、結構比較…………….………30 2-3-9 定點突變 (site-directed mutagenesis) …………………………………31 2-4 討論…………………………………………………………………….……..32 2-4-1岩藻醣激酶及鳥苷二磷酸岩藻醣焦磷酸化酶共同表現之優勢……….32 2-4-2鳥苷二磷酸岩藻醣合成酶的晶體培養…………………………..….….33 2-4-3鳥苷二磷酸岩藻醣合成酶兩區段蛋白之可能構形……………………34 Chapter 3 結果與討論：人類第二型岩藻醣水解酶……………….…….………36 3-1 人類第二型岩藻醣水解酶在大腸菌桿菌之表現及純化…………………...36 3-1-1 以麥芽糖結合蛋白輔助人類第二型岩藻醣水解酶在大腸菌桿菌表現…….36 3-1-2 以不同大腸桿菌菌株及質體篩選解決可溶性聚集問題……………...37 3-2 人類第二型岩藻醣水解酶在畢赤酵母 (P. pastoris) 的表現及純化……...37 3-2-1 質體建構………………...………………………………………………38 3-2-2基因轉殖………………...……………………………………………….38 3-2-3 蛋白質表現量篩選………………...……………………………………39 3-2-4 蛋白質之大量表現量及純化………………...…………………………39 3-3 人類第二型岩藻醣水解酶之生化特性鑑定……...…………………………40 3-3-1第一、二型岩藻醣水解酶序列比對……...…………………………….41 3-3-2人類第二型岩藻醣水解酶之酵素動力學探討……...…………….……41 3-3-3 不同物種表現之FUCA1及FUCA2的生化特性比較…………………42 3-3-4 人類第二型岩藻醣水解酶在畢赤酵母菌表現之醣基化現象………...43 3-4檢測人類岩藻醣水解酶的受質專一性……...………………………………44 3-4-1以標準品為受質………………...……………………………………….45 3-4-2以細胞萃取之醣鏈為受質………………...……………………………..46 3-5 討論……………………………………………………………………………..48 3-5-1 P. pastoris之醣基化特徵………………...……………..……………….48 3-5-2人類岩藻醣水解酶的受質專一性偵測………………....……………........49 Chapter 4 結論……………………………………..……………….…….……….50 4-1 鳥苷二磷酸合成酶……...……………………………………………………50 4-2 人類二型岩藻醣水解酶……...……………………………………………....52 Chapter 5 材料與方法……………………………..…………………..…….…...54 5-1 鬆脆桿菌鳥苷酸合成酶重組蛋白之基因轉殖…………………………...…54 5-1-1 鬆脆桿菌鳥苷酸合成酶之基因取得………………………………...…54 5-1-2 引子設計 (Primer design) ………………………….…………….….…54 5-1-3 聚合酶鏈鎖反應 (Polymerase Chain Reaction，PCR) …………….…..55 5-1-4 黏合反應 (Ligation) ………………………….……………….………..56 5-1-5 定點突變 (site-directed mutagenesis) ………………………….………57 5-1-6 轉殖作用 (Transformation) ………………………….…………………58 5-1-7 質體DNA之抽取………………………….……………….……………58 5-2鬆脆桿菌鳥苷酸合成酶之表現及純化………………………….…………...58 5-2-1 重組蛋白之誘導與大量表現………………………….………………..58 5-2-2 硒代蛋胺酸標定之蛋白質表現 (Se-MET FKP) …………….………...59 5-2-3 重組蛋白之純化………………………….……………….…………….59 5-2-4蛋白質定量………………………….…………………………….……..60 5-3重組脆腸桿菌鳥苷酸岩藻醣合成酶之特性及活性鑑定…………….……...61 5-3-1以TLC片追蹤活性………………………….…………………………..61 5-3-2 酵素動力學參數計算………………………….……………….……….62 5-3-3岩藻醣激酶酵素動力學參數偵測………………………….……………62 5-3-4鳥苷二磷酸化酶焦磷酸化酶酵素動力學參數偵測…………….……....64 5-3-5鳥苷二磷酸岩藻醣合成酶對醣類之受質專一性…………….……...…65 5-3-6 熱穩定度探討………………………….…………………………….….66 5-3-7 分析型超高速離心 (Analytical ultracentrifugation) …………….…....66 5-4重組脆腸桿菌鳥苷酸岩藻醣合成酶之X光晶體學…………….…………..67 5-4-1 養晶條件之篩選(screening)與觀察…………….……………….……...67 5-4-2 養晶條件之優化 (optimization)及培養…………….……………….…67 5-4-3 浸泡受質進入晶體 (soaking) …………….……………….…………...67 5-4-4 X光繞射實驗及數據收集及處理…………….……………….………68 5-4-5 解決相位角問題 (phase problem) …………….……………….………68 5-4-6 結構之建立及精緻化 (refinement) …………….……………….……..68 5-4-7 蛋白質結構圖形之作圖…………….……………….……………….…69 5-5人類第二型岩藻醣水解酶重組蛋白之質體建構…………….……………..70 5-5-1 人類第二型岩藻醣水解酶基因之取得…………….……………….….70 5-5-2 引子設計 (Primer design) …………….……………….……………….70 5-5-3 質體建構…………….……………….……………….…………………71 5-5-4質體的大量製備 …………….……………….……………….…………71 5-6人類岩藻醣水解酶在E.coli的表現與純化…………….……………….……71 5-6-1 人類第二型岩藻醣水解酶 (MBP_FUCA2) 之大量表現…………….71 5-6-2人類第二型岩藻醣水解酶 (MBP_FUCA2) 之純化…………….…….72 5-6-3 人類第一型岩藻醣水解酶 (FUCA1) 重組蛋白之表現與純化 ……..73 5-7 人類第二型岩藻醣水解酶在P. pastoris的表現與純化…………….………73 5-7-1 轉殖質體的準備…………….……………….……………….…………73 5-7-2 勝任細胞的製作…………….……………….……………….…………74 5-7-3 質體轉殖…………….……………….……………….…………………74 5-7-4 蛋白質表現量小量培養及篩選…………….……………….……….…75 5-7-5 蛋白質大量表現及純化…………….……………….……………….…75 5-7-6 蛋白質身分鑑定 (西方墨點法及質譜樣品前處理)…………………..76 5-8 岩藻醣水解酶重組蛋白之活性測定…………….……………….…………77 5-9 岩藻醣水解酶之酵素動力分析…………….……………….………………78 5-10 P. pastoris表現之人類第二型岩藻醣的醣基化分析………………………78 5-10-1 利用PNGase F 切除N-linked glycans……………………………….78 5-10-2 利用質譜分析醣基化位置…………….……………….……………...79 5-11 利用蛋白質變性劑Guanidine hydrochloride萃取細胞的醣蛋白…….…..79 5-11-1 去除脂質…………….……………….………………………………...79 5-11-2 醣蛋白萃取…………….……………….……………………………...79 5-11-3 N-linked glycan釋出與純化…………….……………….…………….80 5-11-4 O-linked glycan釋出與純化…………….……………….………….…81 5-11-5 以質譜儀分析glycan的樣本前處理 (Permethylation)………………81 5-12 P. pastoris 使用之培養液成分配置…………….……………….…….……82 Chapter 6 參考資料……………………………..……………….…….………..117 | |
| dc.language.iso | zh-TW | |
| dc.subject | 岩藻醣 | zh_TW |
| dc.subject | 第二型岩藻醣水解? | zh_TW |
| dc.subject | 鳥?二磷酸合成? | zh_TW |
| dc.subject | 畢赤酵母菌 | zh_TW |
| dc.subject | X 光結晶學 | zh_TW |
| dc.subject | GDP-fucose synthetase | en |
| dc.subject | L-fucose | en |
| dc.subject | X-ray crystallography | en |
| dc.subject | Pichia. Pastoris | en |
| dc.subject | α-L-fucosidase 2 | en |
| dc.title | 鬆脆桿菌鳥苷二磷酸岩藻醣合成酶及人類第二型岩藻醣水解酶之性質探討 | zh_TW |
| dc.title | Characterizations of B. fragilis GDP-fucose synthetase
and Human α-L-Fucosidase 2 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王惠鈞,邱繼輝,梁博煌 | |
| dc.subject.keyword | 岩藻醣,第二型岩藻醣水解?,鳥?二磷酸合成?,畢赤酵母菌,X 光結晶學, | zh_TW |
| dc.subject.keyword | L-fucose,α-L-fucosidase 2,GDP-fucose synthetase,Pichia. Pastoris,X-ray crystallography, | en |
| dc.relation.page | 129 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-08 | |
| 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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