人類第二型岩藻醣水解酶之生化性質探討及偵測

Characterization and Detection of Human α-L-Fucosidase 2

羅鈞(Chun Lo)

指導教授 ： 林俊宏

國立臺灣大學/生命科學院/生化科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10506

摘要

諸多疾病伴隨岩藻醣水解酶活性異常，先前研究發現感染幽門螺旋桿菌會促使人類癌症細胞分泌第二型岩藻醣水解酶 (FUCA2) 至胞外，造成致病菌攝取宿主之岩藻醣。但第二型岩藻醣水解酶在感染過程中扮演之角色與生化性質目前仍不清楚。
本研究為了解第二型岩藻醣水解酶的生化性質及其在幽門螺旋桿菌感染時扮演之角色，首先在人類 293T 細胞表現出有活性的第二型岩藻醣水解酶，檢測其 pH 值對活性之影響 (pH profile)、酵素動力學常數、基質專一性等性質，並與第一型岩藻醣水解酶 (FUCA1) 作比較。這二個酵素的 pH profile 同樣有二個最佳活性區，分別在 pH 4.5 及 pH 6.5。接著用七種含螢光的醣分子檢測基質專一性，發現第一型岩藻醣水解酶可水解 Fuc α-1,2 Gal β-1,4、core Fuc α-1,6 biantennary、Lea、H type I 四種基質上的岩藻醣，而第二型岩藻醣水解酶則水解 Fuc α-1,2 Gal β-1,4、core Fuc α-1,6 biantennary 二種基質的岩藻醣。後者與由人類癌症細胞與幽門螺旋桿菌共同培養液中取得的酵素混合物，作出的結果一致。
另外在幽門螺旋桿菌感染前後的岩藻醣水解酶變化的研究中，利用含螢光基團的岩藻醣水解酶活性分子探針 (ABP) 偵測，發現致病菌感染不僅使胞外培養液中岩藻醣水解酶量及活性上升，也使胞內岩藻醣水解酶分布及活性增加。
綜合以上研究，推測幽門螺旋桿菌感染是會使細胞內岩藻醣水解酶表現增加，進而分泌至胞外。而第二型岩藻醣水解酶在感染時會幫助幽門螺旋桿菌獲取宿主細胞的岩藻醣，且不破壞幽門螺旋桿菌附著與偽裝宿主之醣結構。

關鍵字

人類第二型岩藻醣水解酶幽門螺旋桿菌 ； 基質專一性 ； 活性分子探針

並列摘要

Abnormal fucosidase activity has been associated with many diseases, such as hepatocellular cancer and breast cancer. Previous study indicated that FUCA2 is released by gastric epithelial cells upon Helicobacter pylori infection, and thus a potential diagnostic marker for gastric diseases. Although the enzyme is found to be critical to the bacterial growth, infection, adhesion and pathogenecity, its role and physiological function still remain ambiguous.
　　In this thesis study, the stable lines of recombinant FUCA2 were constructed to determine the biochemical features of FUCA2 such as pH profile, kinetic parameters and substrate specificity. The results indicated that FUCA2 is optimally active at pH 4.5 and pH 6.5, similar to FUCA1. Via the use of seven pyridylamino-conjugated sugar substrates, the HPLC analysis indicates that FUCA1 preferentially hydrolyzes L-fucose from H type II, Lewis antigen A, H type I, and core fucose in the biantennary, while FUCA2 will catalyze the hydrolysis of the first and the last substrates only. Additionally, the fluorescent, activity-based probes LCL10002 and LCL10003, prepared by Prof. L. C. Lo and coworkers in NTU, were studied to detect α-L-fucosidase activity in the dot blot analysis and fluorescent microscopy. The results indicated that LCL10003 is better than LCL10002 in terms of sensitivity, and that both secreted and intracellular fucosidase activities increase after H. pylori infection.

並列關鍵字

α-L-fucosidase ； H. pylori ； substrate specificity ； avtivity-based probe

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