人類岩藻醣水解酶之生化性質探討

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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 , Masters　　Advisor：林俊宏　　

繁體中文

岩藻醣水解酶受質專一性；肝癌；腫瘤指標； α-L-fucosidases ； substrate specificity ； liver cancer ； biomarker

Abstract │ Reference (113) │ Altmetrics

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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.

Reference ( 113 ) 〈TOP〉

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5 Ma, B., Simala-Grant, J. L. & Taylor, D. E. Fucosylation in prokaryotes and eukaryotes. Glycobiology 16, 158R-184R,(2006).

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