探討胜肽基質序列之改變對靈敏螢光分析中酵素專一性之影響

Examining the Effects of Modulation of Peptide Substrate Sequence on the Enzyme Specificity in the Sensitive fluoresence Assay

陳昭隆(Chao-Long Chen)

指導教授 ： 王勝仕

國立臺灣大學/工學院/化學工程學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02211

摘要

摘要

類澱粉b蛋白質(Ab)是一種生理上的胜肽，由類澱粉前驅蛋白(APP)經一系列代謝途徑所生成。釋出胞外的Ab會被許多的內切胜肽酶進行降解代謝，若在腦中的生成與代謝之失衡，則會導致聚集的產生，此為阿茲海默症的成因。阿茲海默症為一種神經退化性疾病，由此可見其發病機制中，Ab的生成是個十分重要的因素，清除新生成的Ab胜肽被認為是治療阿茲海默症可行的策略之一，而可清除Ab胜肽的酵素計有腦啡肽酶 (neprilysin、NEP)、胰島素降解酶 (insulin degrading enzyme、IDE)、內皮素轉化酶 (endothelin converting enzyme、ECE-1)、血管緊張素轉換酶 (angiotensin-converting enzyme、ACE)、血纖維蛋白溶酶(plasmin)、和基質金屬蛋白酶 (matrix metalloproteinase、MMPs)等。
於此研究中，我們合成淬滅螢光之胜肽受質來偵測各種酵素的活性及靈敏度，最初所採用的胜肽是Ab上第十二個胺基酸殘基至第十八個胺基酸殘基，且在最後一個胺基酸殘基後再接上一個半胱胺酸(cysteine, Cys)。一螢光分子連接在胜肽的C端，其發出的螢光會被存在於N端半胱氨酸側鏈上的一淬滅分子所消滅，此基質命名為qf-Ab(12-18)C，當此胜肽基質被酵素水解後，便會放出螢光。
本研究發現共有四種酵素可水解此基質qf-Ab(12-18)C，分別是NEP、ACE、ECE-1與IDE，前兩者活性較高，後兩者反應性較小，沒有明顯的專一性。然後我們嘗試把原本的7個胺基酸殘基的後面兩個殘基改成丙胺酸，經由一樣的合成步驟，得到的新基質命名為qf-Ab(12-16)AAC。本研究發現只剩前兩者NEP與ACE對此基質有活性。明顯地，相較於原本的qf-Ab(12-18)C，qf-Ab(12-16)AAC雖然無法用來偵測單一酵素的活性，但其專一性也有所提升。
此外，在使用細胞進行檢測之方面，有研究指出qf-Ab(1-7)C只能被NEP及IDE水解，而吾人的qf-Ab(12-16)AAC，只能被NEP及ACE水解。利用前述特性，將可建立一互相比較的快速藥物篩檢系統，用以篩檢出對於NEP、ACE、或IDE有增加活性或抑制效果之藥物。
關鍵字：類澱粉b蛋白質、Ab降解酶、螢光共振能量轉移。

關鍵字

類澱粉beta蛋白質 ； A-beta降解酶螢光共振能量轉移

並列摘要

Abstract

Amyloid-b (Ab) is a physiological peptide, which is produced from amyloid precursor protein (APP) by sequential cleavages, and then released into the extracellular spaces. The released Ab undergoes proteolytic degradation by multiple endopeptidases. The imbalance between the production and catabolism of Ab in the brain results in the accumulation of Ab leading to Alzheimer’s disease. Alzheimer’s disease is a neurodegenerative disease, and Ab in Alzheimer’s disease plays a pivotal role in the pathogenesis of the disease. Reduction of Ab production is considered a feasible way in the therapeutics for Alzheimer’s disease. The enzymes that are capable of hydrolyzing Ab include neprilysin (NEP), insulin degrading enzyme (IDE), endothelin converting enzyme (ECE-1), angiotensin-converting enzyme (ACE), plasmin and matrix metalloproteinase (MMPs).
In this study, we synthesized a quenched fluorogenic peptide substrate whose seven-residue sequence was adopted from the twelfth to eighteenth residues of Ab peptide with a cystenine residue (Cys) added at its C-terminal. The fluorescence emission contributed by the fluorophore attached to the C-terminal Cys residue was quenched by the presence of the quencher linked to the N-terminus of the peptide. The synthetic peptide substrate mentioned above was named as qf-Ab(12-18)C. The fluorescence was emitted when this peptide substrate was degraded by enzymes.
We observed in our study that four enzymes exhibit activity toward our synthetic peptide substrate qf-Ab(12-18)C, including NEP, ACE, IDE, and ECE-1. Our results showed that the former two have higher activities, whereas the latter two have lower activities, suggesting that qf-Ab(12-18)C lacks a good enzyme specificity.
Next, we made an attempt to modify the sequence of the peptide substrate, changing the last two residues from luecine and valine to two alanine. Following the same method mentioned previously, a new peptide substrate was obtained and called qf-Ab(12-16)AAC. Only two enzymes, including NEP and ACE, were found to be effective in hydrolyzing the new substrate qf-Ab(12-16)AAC. It is evident that the new peptide substrate qf-Ab(12-16)AAC possesses a superior enzyme specificity over qf-Ab(12-18)C.
Moreover, the cell-based assay was performed in our study. Our previous investigation showed that qf-Ab(1-7)C can be hydrolyzed by only two enzymes, NEP and IDE. However, we found in this work that NEP and ACE are able to hydrolyze qf-Ab(12-16)AAC. We believe the combination of these two different peptide substrates allows us to develop a fluorescence-based platform for detecting the chemicals which can enhance or reduce the activity of important Ab-degarding enzymes.

Key words：Amyloid-b, Ab-degrading enzymes, neprilysin, angiotensin-converting enzyme, insulin degrading enzyme, fluorescence resonance energy transfer (FRET).

並列關鍵字

Amyloid-beta ； A-beta-degrading enzymes ； neprilysin ； angiotensin-converting enzyme ； insulin degrading enzyme ； fluorescence resonance energy transfer

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