人類岩藻醣水解酶之生化性質探討

Characterization of Human α-L-Fucosidase: Large-scale preparation, substrate specificity

高敏絹(Min-Chuan Kao)

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(2012年)

https://doi.org/10.6342/NTU.2012.10388

摘要

岩藻醣水解酶 (α-L-Fucosidase) 是負責水解非還原端上各種含有岩藻糖修飾的醣類分子並釋放出L-fucose。人類有兩種岩藻醣水解酶 (EC 3.2.1.51) ，分別為第一型岩藻醣水解酶 (簡稱FucA1) 和第二型岩藻醣水解酶 (簡稱FucA2) 。諸多疾病會有岩藻醣水解酶活性異常的現象，先前本實驗室的研究發現，當人類癌症細胞受到胃幽門螺旋桿菌感染時，會促使FucA2分泌至細胞外。而FucA2在感染過程中所扮演的角色與生化性質目前仍不清楚。
為了瞭解FucA2的生理意義，探討其生化特性是很重要的一部份。本研究首次在大腸桿菌建構具有融合蛋白的FucA2質體，並成功表現出有活性且大量的FucA2，檢測其pH值對活性之影響、酵素動力學常數、受質專一性等性質，並與FucA1作比較。這二個酵素的pH 值活性分布有異，FucA1最佳活性落在pH 4.5 及pH 6.5而FucA2則落在pH 6.0。接著用含螢光的寡醣鏈與細胞萃取之醣鏈檢測受質專一性，發現FucA1可水解 Fuc α-1,2 Gal β-1,4、core Fuc α-1,6 bi/triantennary、Lea、Lex受質上的岩藻醣，而且FucA1對於細胞上萃取之後的醣鏈上之core Fucose有非常好的水解活性，FucA2則水解Fuc α-1,2 Gal β-1,4、core Fuc biantennary、Lex受質上的岩藻醣。
另外諸多研究報告癌症病人的血漿中含有高度的岩藻醣水解酶活性，為探究此生理現象的活性來源，本研究自萬芳醫院取得癌症病人的血漿進行分析，並利用實驗室合成的抑制劑層析管柱純化出岩藻醣水解酶，再以中研院單多株抗體生產中心所生產的專一性抗體來作確認。經實驗證實肝癌病人血漿中高度的岩藻醣水解酶活性來自於FucA1，而且發現其上具有轉譯後修飾作用，以凝集素微陣列分析發現該修飾為含有Fuc之N-glycosylation，而正常人血漿中岩藻醣水解酶則屬於FucA2，其修飾則偏向O-glycosylation。顯示FucA1有潛力作為腫瘤指標，而單多株抗體生產中心所製備的anti-FucA1 IgG則可應用於臨床研究並作為診斷工具。

關鍵字

岩藻醣水解酶受質專一性 ； 肝癌 ； 腫瘤指標

並列摘要

There are two α-L-fucosidases (EC 3.2.1.51) in human, including α-L-fucosidase1 (FucA1) and α-L-fucosidase2 (FucA2). They are responsible for the removal of L-fucose residues from the non-reducing end of glycoconjugates. Abnormal fucosidase activity has been associated with many diseases, such as hepatocellular cancer and breast cancer. Previous study indicated that the infection of Helicobacter pylori triggers the release of FucA2 by gastric cancer cells. However, the role and physiological function of FucA2 still remain ambiguous. One of the prerequisites for further studies is to prepare FucA2 in a sufficient amount and to carry out their characterizations.
Previous preparation of FucA2 in our labotoary was restricted by the poor expression and extremely low activity. Herein we succesfully express recombinant FucA2 in E. coli.by incorporating 6x His, MBP and TEV protease recognition site to N-terminus of FucA2. Significant expression and activity were obtained after removal of the MBP-fusion tag with TEV protease, allowing the biochemical characterizations such as pH profile, kinetic parameters and substrate specificity. The results indicated that FucA2 is optimally active at pH 6.0, unlike FucA1 which has dual optimum at pH 4.5 and pH 6.5.
To examine the substrate specificity, the glycoproteins extracted from human colon cancer cells line (Colo 205) and seven pyridylamino-conjugated sugars were used for the study. These glycoconjugates were subjected to the hydrolytic cleavage of either FucA1 or FucA2, followed by mass spectrometric and HPLC analyse, respectively the results indicate that FucA1 preferentially hydrolyzes L-fucose from Fuc α-1,2 Gal β-1,4, core Fuc α-1,6 bi/triantennary, Lea, Lex, while FUCA2 catalyzes the hydrolysis of Fuc α-1,2 Gal β-1,4, and Lex.
Furthermore, both recombinant FucA1 and FucA2 were examined by the antibodies that were generated by immunization of the specific peptides correspounding to residues 404-444 of FucA1 and residues 404-445 of FucA2 (represent the major sequence differance). In conjunction with our developed inhibitor-based affinity column chromatography, the antibodies were used to examine and identify the serum fucosidase from the patients with liver and gastric cancers. The result indicated that high activity of FucA1 was found in the serum of liver cancer patients, while that of FucA2 in the serum of normal people. Further examination (such as the glycol-structure analysis) would be beneficial to use FucA1 as a biomarker.

並列關鍵字

α-L-fucosidases ； substrate specificity ； liver cancer ； biomarker

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