蛋白质O-GalNAc糖基化抑制剂的分子机制研究

O-GalNAc glycosylation is an important protein modification and its abnormal state is closely related with a variety of disease. ppGalNAc-T is a key enzyme that regulates the initial step of protein O-GalNAc glycosylation. The inhibitor of ppGalNAc-T is an important tool of researching O-GalNAc glycosylation. However, there is no inhibitor of O-GalNAc glycosylation with obvious effect and distinct inhibitory mechanism internationally. In our previous studies, on the basis of enzyme-linked coupling reaction, we developed a type of high-throughput glycosyltransferase inhibitor screening model. Using this model, we screened approximately 5000 natural compounds and finally identified a natural compound which can effectively inhibit enzyme activity of ppGalNAc-T and glycosylation of cell. We also found that this compound can inhibit O-GalNAc glycosylation on amyloid precursor protein(APP), which is the precursor of pathogenesis-related protein of Alzheimer’s disease, and affect its maturation and processing. In this project, by using methods of glycobiology, enzyme biochemistry, cytobiology and animal models study, we will focus on illuminating the inhibitory molecular mechanism of enzyme activity of ppGalNAc-T and functional mechanism of inhibiting O-GalNAc glycosylation on APP of this natural compound and provide new explanation for the pathogenesis and remedy for Alzheimer's disease.

O-GalNAc糖基化是重要的蛋白质修饰，其异常修饰与多种疾病的发生发展密切相关。ppGalNAc-T是蛋白质起始O-GalNAc糖基化的关键调控酶。ppGalNAc-T抑制剂是研究O-GalNAc糖基化的重要工具。目前国际上缺乏抑制效果明显且机理明确的O-GalNAc糖基化抑制剂。我们基于酶联耦合反应原理开发了高通量糖基转移酶抑制剂筛选方法，从约5000种天然化合物中筛选到对抑制ppGalNAc-T酶活和细胞糖基化均有效的化合物分子，该化合物可抑制阿尔兹海默病的病原前体蛋白APP（amyloid precursor protein）的O-GalNAc糖基化并影响其分泌剪切。本项目拟用糖生物学、生物化学、细胞生物学和动物模型实验等方法，系统研究天然化合物小分子抑制剂抑制ppGalNAc-T酶活的分子机制；同时阐明该抑制剂抑制APP糖基化的功能机制，为理解阿尔兹海默病的发病与治疗提供参考。

黏蛋白型O-糖基化（又称O-GalNAc糖基化）是一种重要的蛋白质翻译后修饰类型，参与维持细胞正常生理功能并与多种疾病的发生、发展密切相关。多肽: N-乙酰氨基半乳糖转移酶（ppGalNAc-T）家族有20个成员，是催化蛋白质O-GalNAc糖基化起始的糖基转移酶。ppGalNAc-T酶将供体UDP-N-乙酰氨基半乳糖 (UDP-GalNAc) 的N-乙酰氨基半乳糖基(GalNAc)连接到受体蛋白的丝氨酸或苏氨酸的羟基上形成GalNAc-Ser/Thr初始结构。ppGalNAc-T酶通过对底物的选择性催化精密调控蛋白质的O-GalNAc糖基化。通过小分子抑制剂干预ppGalNAc-T酶的催化活性进而调控底物蛋白的加工、成熟等过程，对研究O-GalNAc糖基化的生物学功能具有重要意义。本研究中，利用基于HPLC的ppGalNAc-T酶活性检测体系，我们发现天然黄酮多酚化合物木犀草素（Luteolin）和单宁多酚化合物鞣花酸（Ellagic acid）和尿石素D（Urolithin D）是ppGalNAc-T酶的有效小分子抑制剂。这些化合物能够直接与ppGalNAc-T酶受体底物结合区结合，并通过与受体多肽/蛋白竞争发挥抑制ppGalNAc-T酶的催化活性。进一步，针对阿尔兹海默症（Alzheimer’s disease, AD）相关的淀粉样前体蛋白（amyloid precursor protein, APP），我们发现黄酮多酚化合物Luteolin 能够选择性调控 ppGalNAc-T酶对APP 的催化活性，并能够通过抑制O-GalNAc 糖基化减少ABeta形成，从而对阿尔兹海默病的症状起到缓解作用。针对单宁多酚化合物，我们发现单宁多酚化合物Urolithin D能够通过抑制肿瘤相关的平足蛋白（podoplanin，PDPN）上的O-GalNAc糖基化修饰，降低PDPN的蛋白稳定性，进而抑制结直肠癌肿瘤细胞的侵袭和迁移。本项目研究成果为糖基转移ppGalNAc-T酶抑制剂的开发提供了重要的先导化合物和分子结构基础，并从功能糖生物学的角度解释天然多酚类化合物治疗疾病的分子机制。同时通过所发现的抑制分子揭示了蛋白质位点特异的糖基化修饰精准调控的重要生物学意义。