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標題: | 探討岩藻醣水解酶應用在岩藻醣化醣類之定序 Identification of fucosidases for sequencing fucosyl glycans |
作者: | Heng-Hsin Lin 林姮昕 |
指導教授: | 翁啟惠 |
關鍵字: | 岩藻醣水解?,岩藻醣,醣類定序, fucosidases,fucose,glycan sequencing, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 醣化作用是一種主要的共同轉譯作用或轉譯後修飾作用,蛋白質醣化作用是一種非模板編碼階段式的調控,包括醣類生成時的延長、分支、終止,這樣的機制導致醣類有極大的異質性,因此醣蛋白會是多種醣型的混和物。而糖化作用對於蛋白質的結構和功能有很大的影響,像是當表皮生長因子受體唾液酸化時,會讓配體誘導表皮生長因子受體形成二聚體的程度下降,或是將抗體上的核心岩藻醣去除,會增加抗體依賴的細胞介導的細胞毒性作用作用。由於醣類生成的過程會造就各式複雜的糖類同分異構物,對於如何解構各醣類的組成和單醣之間的鍵結是一個很大的挑戰,特別是在醣類岩藻醣化的過程,由於岩藻醣可以加在N-醣鏈糖上核心或末端的位置,因而更增加了醣種類的複雜度。而在醣類定序上單獨使用質譜儀並不能完整提供醣類所有資訊,所以我們選擇使外切糖苷酶,利用酵素的專一性,可以得到各單醣之間的鍵結的資訊。在本篇研究中,我們利用研究多種岩藻醣酶的專一性來應用在醣類定序。為此,我們使用市售來源或用化學、生物性合成的醣當作酵素的受質來分析其專一性。
在定量分析每個岩藻醣水解酶的活性方法上,我們使用還原胺化作用,讓醣接上2-胺基苯甲醯,如此一來醣類將會帶上螢光利於在液相色譜上被偵測到。而為了尋找具有良好專一性的岩藻醣水解酶,我們從CAZy 資料庫中挑選並將之純 化出來,並分析純化出的岩藻醣水解酶對於不同醣鏈岩藻醣其專一性及活性,在本研究中,我們將這些新發現的岩藻醣水解酶使用在半乳糖凝集素-3結合蛋白當作一範例,證明利用酵素個別的專一性,可以達到醣類定序之目的。 Glycosylation is the major type of co- and post-translational modification. Protein glycosylation endows with extreme heterogeneity through non-template encoded step-wise elongation, branching and termination. Therefore, each glycoprotein is heterogeneously glycosylated. It has been reported that glycosylation of proteins plays a variety of structural and functional roles, for example, sialylation decreases ligand-induced EGFR dimerization, and removal of core fucose in IgG enhances antibody-dependent cell mediated and complement-dependent cytotoxicity. Due to the complexity of glycans that creates lots of glycan isomers, it is challenging to delineate the exact composition and sequence of glycan chains. Particularly, protein fucosylation that can occur at either the terminal or core position generates the most complicated species of N-linked glycans. To tackle the problem of glycan sequencing that mass spectrometry alone is inadequate to solve, we employed exoglycosidases, which could provide linkage information of glycan chains, if the specificity of the exoglycosidase used is distinct and clear-cut. We chose to focus on exploring the specificities of fucosidases for the purpose of glycan sequencing. To survey for substrate specificities of fucosidases, we first collected many glycan substrates from either commercial sources or glycan synthesis with chemical or enzymatic approaches. To analyze the hydrolysis ability of fucosidases in a quantitative method, we utilized reductive amination to tag glycan substrates with 2-aminobenzamide for the purposes of fluorescent detection and separation with liquid chromatography. In order to look for the fucosidases with good specificities for glycan sequencing, we cloned and purified selected fucosidases from CAZy (Carbohydrate-Active enZymes database) and examined for the enzymetic specificity toward fucoses with different linkages and in different glycan units. Here, we used the galectin-3 binding protein (Gal-3BP) as a model to show that the novel fucosidases with unique substrate specificity could be applied for glycan sequencing. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77694 |
DOI: | 10.6342/NTU201703154 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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