阿茲海默症（Alzheimer’s disease， AD）是最常見的神經退化性疾病，其特徵為進行性認知功能衰退。類澱粉蛋白 β（amyloid-β， Aβ）的生成被認為是 AD 發病的重要病理機制之一，而 Aβ 的產生始於 β-分泌酶 1（β-secretase 1， BACE1）對類澱粉前驅蛋白（amyloid precursor protein， APP）的切割。因此，BACE1 已成為阿茲海默症治療研究中的重要藥物標的。 本研究以 Expi293F 細胞表達人類 BACE1 胞外結構域蛋白，並透過親和層析與膠體過濾層析進行純化。進一步製備並分析不同醣基化型態之 BACE1，包括成熟複合型醣基化（mature complex glycoform， BACE1-C）、高甘露糖型醣基化（high-mannose glycoform， BACE1-HM）以及單醣基化型（mono-glycosylated glycoform，BACE1-MG）。利用螢光共振能量轉移（fluorescence resonance energy transfer， FRET）酵素活性分析系統評估各醣型 BACE1 之酵素活性，並測定其穩態動力學參數，以探討醣基化修飾對 BACE1 催化功能之影響。 此外，本研究亦評估醣類分子化合物對 BACE1 之抑制作用，透過酵素抑制實驗及酵素動力學分析探討其抑制效果，並進一步研究 BACE1 與抑制劑之間的結合特性，以闡明其作用機制。研究結果顯示，BACE1 的醣基化型態會顯著影響其催化效率及對抑制劑的敏感性。上述發現有助於深入理解 BACE1 的結構與功能關係，並為阿茲海默症相關藥物之開發提供理論基礎與研究方向。;Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by progressive cognitive decline. The generation of amyloid-β (Aβ) peptides， a key pathological hallmark of AD， is initiated by the proteolytic cleavage of amyloid precursor protein (APP) by β-secretase 1 (BACE1). Therefore， BACE1 has emerged as an important therapeutic target for AD intervention. In this study， the extracellular domain of human BACE1 was expressed in Expi293F cells and purified using affinity and size-exclusion chromatography. Different glycosylation forms of BACE1， including the mature complex glycoform (BACE1-C)， high-mannose glycoform (BACE1-HM)， and mono-glycosylated glycoform (BACE1-MG)， were prepared and characterized. The enzymatic activities of these glycoforms were evaluated using a fluorescence resonance energy transfer-based assay， and steady-state kinetic parameters were determined to investigate the influence of glycosylation on catalytic function. Furthermore， The inhibitory effects of selected synthetic heparan sulfate (HS) oligosaccharides on BACE1 were evaluated using enzyme inhibition assays and enzyme kinetic analyses. Binding interactions between BACE1 and inhibitors were also investigated using bio-layer interferometry to elucidate their inhibitory mechanisms. The results demonstrated that glycosylation significantly affects the catalytic efficiency and inhibitor sensitivity of BACE1. These findings provide insights into the structure–function relationship of BACE1 and contribute to the development of novel therapeutic strategies targeting Alzheimer’s disease.
