探討N-神經醯胺水解酶2 (ASAH2)第二亞型(Isoform 2) 的功能與結構

Unveiling the Function and Structure of N-Acylsphingosine Amidohydrolase 2 (ASAH2) Isoform 2

劉思慧(Shi Hui Law)

指導教授 ： 柯良胤

高雄醫學大學/健康科學院/醫學檢驗生物技術學系碩士班/碩士(2019年)

摘要

人體在正常情況下，血液中的溶血卵磷脂(LPCs)濃度都維持在125 to 143 nmole/mL。血漿內高濃度的LPCs會促進發炎因子如介白素-1β (IL-1β)、介白素-6 (IL-6)、α型腫瘤壞死因子(TNF-α)、γ型干擾素(IFN-γ)的釋放、B細胞及巨噬細胞的活化，進而引起動脈粥狀硬化前期最重要的發炎反應。此外，在腦組織中LPCs濃度的上升也會造成外被細胞(pericyte)受損、脫鞘作用(demyelination)以及澱粉樣蛋白(amyloid)??1-42的堆積，進而導致神經退化性疾病。已知目前體內的LPCs會被具有lysoPLD活性的酵素(autotaxin) 代謝成更具生物毒性的溶血磷脂酸(LPA)，與癌症的發生有關；若有lysoPLA1活性的酵素則可將LPCs代謝成對人體無害的產物，然而目前都沒有專一執行lysoPLA1活性的酵素。利用液相層析法/質譜儀技術對脂質及其代謝物的分析，我們發現了一個可執行lysoPLA1活性的酵素(ASAH2)。為證明此重要的發現，我們以分子模擬(molecular docking and simulation)的方式預測出重要的酵素胺基酸活化位。藉由位點導向基因突變(site-direct mutagenesis)技術製作出有基因突變的ASAH2，將用於評估酵素活化位及驗證酵素活性。我們的研究結果顯示：以ASAH2與脂質標準品作用，在2小時之內可清除97%的LPCs。另外，陰電性低密度脂蛋白(L5-LDL)是人體內大量攜帶LPCs的物質，將L5-LDL與ASAH2反應2小時可以降低60%的LPCs。根據分子模擬預測結果，我們製作出突變ASAH2-T425A、Y544A及Y556A，突變後代謝LPCs的酵素活性功能降低。我們的研究揭露了ASAH2的酵素功能，而lysoPLA1酵素活性的氨基酸位點為T425A、Y544A及Y556A。利用此新型酵素來分解LPCs，未來將可作為一個有效的治療心血管疾病的方法。

關鍵字

溶血卵磷脂(LPCs) ； 心血管疾病 ； 溶血磷脂酸 (LPA) ； ASAH2

並列摘要

Lysophosphatidylcholines (LPC) is a class of lipid biomolecule derived by the cleaving of phosphatidylcholine (PC) via the action of phospholipase A2 (PLA2) or by the transfer of fatty acids to free cholesterol via lecithin-cholesterol acyltransferase (LCAT). In healthy individuals, the plasma level of LPC ranges from 125 to 143 nmole/mL, but its levels increase in diseases such as cardiovascular diseases (CVDs), diabetes, ovarian cancer, and renal failure. Although LPCs will be produced in the circulation when PLA2 cleaves PCs, they can be converted back to PCs by the enzyme lysophosphatidylcholine acyltransferase (LPCAT) in the presence of Acyl-CoA. These two pathways built up the Lands cycle which already be known in previous researches. We found that accumulation of LPCs in circulation also can be degraded by autotaxin (ATX), an enzyme with lysoPLD activity, to become more harmful metabolites lysophosphatidic acid (LPA) which associated with cancer. Accidentally, we found an enzyme N-acylsphingosine amidohydrolase 2 (ASAH2), a kind of ceramidase which not only capable of hydrolyzing ceramide (CER) but also can hydrolyze LPC by using lysophospholipase A1 (lysoPLA1) activity. By the observation, we aim to revealed lysoPLA1 activity of ASAH2 which beyond Lands cycle and capable of degrading both the plasma CERs and LPCs into harmless metabolites. Our previous results showed that ASAH2 was capable to degrade up to 97% of LPC standards within 2 hours. Besides, ASAH2 also able to degraded about 60% of LPCs in L5-LDL. The domains T425, Y544 and Y556 are required for carrying out lysoPLA1 activity of ASAH2. Due to the unexpected findings, a novel enzymatic function of ASAH2 for degrading LPCs may be a potential therapeutic to prevent and ameliorate cardiovascular diseases in the future.

並列關鍵字

Lysophosphatidylcholine (LPC) ； cardiovascular diseases (CVDs) ； lysophosphatidic acid (LPA) ； N-acylsphingosine amidohydrolase 2 (ASAH2)

參考文獻

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