溶酶體儲積症（lysosomal storage diseases， LSDs）是一類由溶酶體酵素缺乏或功能異常所引起的遺傳性疾病，導致未被降解的代謝物在細胞內累積。酵素替代療法（enzyme replacement therapy， ERT）目前是治療溶酶體儲積症的重要策略之一。然而，許多重組溶酶體酵素的甘露糖-6-磷酸（mannose-6-phosphate， M6P）修飾程度不足，因而降低其被甘露糖-6-磷酸受體（mannose-6-phosphate receptor， M6PR）辨識的能力，進一步限制酵素的細胞攝取及溶酶體遞送效率。因此，增強重組酵素與M6PR之間的辨識能力，是提升酵素替代療法治療效率的重要策略。 本研究以與α-甘露糖苷儲積症（α-mannosidosis）相關的溶酶體酵素α-甘露糖苷酶（α-mannosidase， α-hLM）作為模式蛋白，建立一套透過化學方式嫁接M6P配體以提升細胞攝取效率的策略。首先，選用6-O-磷酸化的α(1，2)-連結甘露糖雙醣作為M6PR標靶配體的核心結構，並設計及合成具有不同聚乙二醇（polyethylene glycol， PEG）連接鏈長度與配體價數的M6P配體。接著，分別透過離胺酸定向嫁接及唾液酸氧化介導嫁接，將M6P配體修飾至由293F細胞表現的α-hLM上，以探討嫁接位置、配體價數及連接鏈長度對細胞攝取效率的影響。 此外，本研究使用α-甘露糖苷儲積症細胞，評估M6P修飾α-hLM的細胞攝取能力及細胞內酵素活性。利用液相層析串聯質譜法（liquid chromatography–tandem mass spectrometry， LC–MS/MS）分析細胞內富含甘露糖之寡醣的累積變化，以初步評估修飾後蛋白是否能改善異常的醣類代謝。同時，使用高效能陰離子交換層析搭配脈衝安培偵測法（high-performance anion-exchange chromatography with pulsed amperometric detection， HPAEC–PAD）分析蛋白質的M6P含量，以確認此化學嫁接策略是否能提高α-hLM的M6P修飾程度。 本研究所建立的M6P配體化學嫁接策略可提升重組溶酶體酵素的細胞攝取效率，並提供一個可調整配體價數、連接鏈長度及蛋白質嫁接位置的修飾平台。此策略未來具有應用於溶酶體酵素修飾及酵素替代療法最佳化的潛力。;Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal enzyme deficiencies or dysfunction， leading to the accumulation of undegraded metabolites within cells. Enzyme replacement therapy (ERT) is currently an important treatment strategy for LSDs. However， many recombinant lysosomal enzymes suffer from insufficient mannose-6-phosphate (M6P) modification， which reduces their recognition by the mannose-6-phosphate receptor (M6PR)， thereby limiting cellular uptake and lysosomal delivery. Therefore， enhancing the recognition between recombinant enzymes and M6PR is an important approach for improving ERT efficiency. In this study， α-mannosidase (α-hLM)， a lysosomal enzyme associated with α-mannosidosis， was used as a model protein to establish a strategy for enhancing cellular uptake efficiency through chemical conjugation with M6P ligands. First， a 6-O-phosphorylated α(1，2)-linked mannose disaccharide was selected as the core structure of the M6PR-targeting ligand， and M6P ligands with different polyethylene glycol (PEG) linker lengths and ligand valencies were designed and synthesized. Next， the M6P ligands were conjugated to α-hLM expressed in 293F cells through lysine-directed conjugation and sialic acid oxidation-mediated conjugation to investigate the effects of conjugation site， ligand valency， and linker length on cellular uptake efficiency. Furthermore， the cellular uptake capacity and intracellular enzyme activity of M6P-modified α-hLM were evaluated using α-mannosidosis cells. Changes in the accumulation of mannose-rich oligosaccharides were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to preliminarily assess whether the modified protein could improve abnormal carbohydrate metabolism. In addition， high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) was used to analyze the M6P content of the protein and confirm whether the chemical conjugation strategy could increase the M6P modification level of α-hLM. The established M6P ligand chemical conjugation strategy can improve the cellular uptake efficiency of recombinant lysosomal enzymes and provides a modification platform in which ligand valency， linker length， and protein conjugation sites can be adjusted. This strategy has potential applications in lysosomal enzyme modification and the optimization of enzyme replacement therapy.
