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

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。

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.