人類海藻糖水解酶在大腸桿菌中的功能性表現與新型海藻糖水解酶抑制劑的檢測 Functional expression of human trehalase in Escherichia coli and identification of novel trehalase inhibitors

Date
2013
Authors
黃胤榮
Yin-Jung Huang
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
海藻糖水解酶、蔗糖酶-異麥芽糖 (sucrase-isomaltase, SI)、麥芽糖酶-葡萄糖澱粉酶 (maltase-glucoamylase, MG) 是哺乳類小腸絨毛膜主要的α-葡萄糖苷酶 (α-glucosidases),這些酵素負責把雙醣水解成單醣,以利吸收作為能量的來源。海藻糖存在於很多食物當中,而小腸海藻糖水解酶 (EC 3.2.1.28) 會水解海藻糖 (1-α-D-glucopyranosyl α-D-glucopyranoside) 成為兩個葡萄糖分子。海藻糖對細胞有兩種保護的特性,亦即可當作化學伴護劑 (Chemical chaperone) 及細胞自噬反應引發劑 (inducer of autophagy),實驗證實在神經細胞當中是可以運用在因蛋白質沉澱所造成的退化性神經疾病的藥物治療上。因此,如果能透過抑制腸道內的海藻糖水解酶活性,進而增加腸道內海藻糖吸收的話,這樣在血液和大腦中的海藻糖濃度也會增加,有可能改善由蛋白質沉澱所造成的退化性神經疾病。而人類海藻糖水解酶的結構、催化機制與其抑制劑到目前為止還沒有被研究清楚,本研究將人類海藻糖水解酶的cDNA在大腸桿菌裡表達,然而表達出來的重組蛋白質為內聚體 (inclusion body),利用透析式摺疊 (Dialysis refolding) 和管柱色層層析摺疊法 (On-column refolding) 進行蛋白質再摺疊復性,但是再摺疊出來的重組蛋白質活性非常低。為了避免因蛋白分子間及分子內不正常的雙硫鍵形成而造成蛋白質錯誤的摺疊,以增加它的可溶性,根據三級結構的模擬把人類海藻糖水解酶中可能不涉及形成雙硫鍵的半胱胺酸 (cysteine) 殘基,利用定位突變法以絲氨酸 (serine) 來取代,其中預測出4個彼此距離較遠、較不可能形成雙硫鍵的半胱胺酸,並將它們突變為絲氨酸。然而突變的重組蛋白質仍為內聚體,而且再摺疊出來的重組蛋白質仍然沒有活性。另外,生化分析數種海藻糖的結構類似物對於豬的海藻糖水解酶的抑制作用,確定可以作為哺乳類海藻糖水解酶的抑制劑,這些類似物有可能作為治療由蛋白質沉澱所造成的神經性退化性疾病的藥物。 關鍵字: 海藻糖、海藻糖水解酶、海藻醣水解酶抑制劑、重組蛋白質表達
In mammals, trehalase, sucrase-isomaltase and maltase-glucoamylase are the major α-glycosidases of the intestinal brush border membranes. These enzymes are responsible for the degradation of di- and oligosaccharides into monosaccharides, and are crucial for the energy-intake. Trehalase (EC 3.2.1.28) hydrolyses α,α-trehalose (1-α-D-glucopyranosyl α-D-glucopyranoside) to two glucose molecules. The intestinal trehalase is involvedin the hydrolysis of ingested trehalose which is found mainly in many nutrient foods. The dual protective properties of trehalose (as a chemical chaperone and an inducer of autophagy) have encouraged pharmaceutical application of the disaccharide in neurodegenerative diseases caused by protein aggregation process. Therefore, it is theoretically possible to increase intestinal absorption of trehalose through inhibiting intestinal trehalase activity, and thus increase in trehalose content in blood or brain. This may in turn alleviate neurological protein deposition diseases. The protein structure, catalytic mechanism and specific inhibitors of human intestinal trehalase (hTreH) have not been elucidated. In the present study, a cDNA fragment encoding the mature form of hTreH was cloned and recombinant hTreH was expressed in Escherichia coli. However,the recombinant hTreH was expressed as an inclusion body. Protein refolding through dialysis and on-column refolding process were performed. The refolded enzyme showed very low specific activity. To prevent protein misfolding through the formation of incorrect intra- or inter-molecular disulfide bonds and thus increase its solubility, based on tertiary structure modeling, several predicted non-disulfide-bonding cysteine residues in hTreH were replaced with serine by site-directed mutagenesis. Four cysteine residues in hTreH were changed into serine, which are predicted to be distant from each other and may not form disulfide bonds with each other. However, the mutant proteins were also expressed as inclusion bodies, and the refolded enzymes still showed no activity. Several trehalose analogs were biochemically characterized as mammalian trehalase inhibitors, and they can be as potential therapeutics for the protein deposition-mediated diseases. Keyword: trehalose, trehalase, trehalase inhibitor, recombinant protein expression
Description
Keywords
海藻糖, 海藻糖水解酶, 海藻醣水解酶抑制劑, 重組蛋白質表達, trehalose, trehalase, trehalase inhibitor, recombinant protein expression
Citation
Collections