β4GalT1调节TNFR1的糖基化修饰在小胶质细胞炎性激活中的作用

Accumulating evidence demonstrated that beta 1,4-galactosyltransferaseⅠ(β4GalT1)-regulated protein glycosylation and the autocrine loop of tumor necrosis factor receptor-α (TNF-α) greatly contribute to microglia activation and the inflammation of central nervous system (CNS). The results of our preliminary experiments suggested that β4GalT1 could bind with TNF-α receptor 1 (TNFR1) and regulate TNFR1 glycosylation in microglia. Based on these findings, we hypothesized that, in microglia, β4GalT1 modulates TNFR1 glycosylation and facilitates its capability of binding with TNF-a, through which β4GalT1 promotes the formation of TNF-α autocrine loop, advances microglia activation and thus participates in the CNS inflammation. To verify this hypothesis, in this project, first, we will study the glycosylation of TNFR1 in mice microglia, and analyze its influence on the protein stability, intracellular transport and ligand binding capability of TNFR1; next, we plan to analyze the interaction between β4GalT1 and TNFR1, and evaluate β4GalT1-mediated TNFR1 glycosylation in microglia; then, we are going to explore the influences of β4GalT1-mediated TNFR1 glycosylation on the TNF-α autocrine loop in microglia; finally, we aim to demonstrate its significance in microglia inflammatory activation and the subsequent neural injury. Based on the above observations, we are going to systematically analyze the biological significance of β4GalT1-regulated TNFR1 glycosylation during microglia activation and CNS inflammation. By this study, we aim to provide some valuable experimental evidence for a better understanding of the significance of protein glycosylation in microglia inflammatory activation and CNS disease.

β-1,4-半乳糖基转移酶-1（β4GalT1）调控的蛋白质糖基化修饰及肿瘤坏死因子-α（TNF-α）自分泌循环在小胶质细胞激活和中枢神经系统（CNS）炎症反应中发挥重要作用。课题组前期发现β4GalT1可调节肿瘤坏死因子受体1（TNFR1）的糖基化修饰，据此提出β4GalT1通过调控TNFR1糖基化修饰，影响其与TNF-α的结合，参与TNF-α自分泌循环，调节小胶质细胞炎性激活和CNS炎症反应。课题拟首先明确TNFR1的糖基化修饰、分析其对TNFR1配受体结合能力、胞内转运及蛋白稳定性的影响；接着分析β4GalT1对TNFR1糖基化修饰的调控机制；在此基础上明确β4GalT1调节TNFR1糖基化修饰对小胶质细胞TNF-α自分泌循环的影响；最后分析上述修饰调节在小胶质细胞炎性激活及继发性神经元炎性损伤中的作用。本研究为阐明蛋白质糖基化修饰在CNS疾病中的作用提供实验依据。

小胶质细胞炎性激活广泛参与中枢神经系统疾病发生发展。蛋白质糖基化修饰是重要的蛋白质翻译后修饰机制，调节蛋白质折叠、加工、成熟、配受体结合、信号转导等，与炎症反应密切相关。β1,4-半乳糖基转移酶-1（beta 1,4-galactosyltransferase Ⅰ，β4GalT1）是最早被克隆的糖基转移酶之一，参与调节机体免疫应答。肿瘤坏死因子受体1（tumor necrosis factor receptor-1，TNFR1）广泛表达于多种细胞（包括小胶质细胞）表面，是TNF-α发挥促炎生物学活性的主要受体，但其糖基化修饰鲜有研究。本课题以小胶质细胞为研究对象，以蛋白质糖基化修饰为切入点，采用分子生物学、细胞生物学、糖生物学等技术，探讨β4GalT1调控TNFR1糖基化修饰对小胶质细胞炎性激活的调节作用及机制。.课题研究发现：小胶质细胞TNFR1蛋白存在N-糖基化修饰，其位点为Asn 151及Asn 202；TNFR1的N-糖基化修饰不影响其蛋白表达及亚细胞定位，但有助于其与配体TNF-ɑ结合；β4GalT1可与TNFR1结合形成复合物，催化TNFR1的Galβ1-4GlcNAc糖链形成，该糖基化修饰随小胶质细胞炎性活化而进一步增强；β4GalT1对TNFR1的糖基化修饰可增强其与配体TNF-α的结合能力，促进小胶质细胞TNF-α自分泌循环；β4GalT1对TNFR1的糖基化修饰可促进TNF-α诱导的NF-κB信号通路活化，促进诱导型一氧化氮合酶（iNOS）表达、活性氧（ROS）生成，从而促进小胶质细胞炎性活化。课题还分析了蛋白质糖基化修饰与其他炎性疾病间的关系，发现转录因子NF-κB的O-GlcNAc糖基化修饰参与胰腺腺泡细胞炎症反应；β4GalT1调节趋化因子受体CXCR3的N-糖基化修饰，促进成纤维样滑膜细胞侵袭，参与类风湿性关节炎；TRAF6-p38/JNK-ATF2 轴可促进小胶质细胞炎性活化。 .课题研究结果有助于进一步阐明蛋白质糖基化修饰在神经炎症相关疾病中的作用及机制，并为相应疾病临床诊疗提供理论依据及新的潜在分子靶点。