Core Fucosylation of N-linked Glycans as a Regulator of CD8+ T cell Activation and Function

N 连接聚糖的核心岩藻糖基化作为 CD8 T 细胞激活和功能的调节剂

• 批准号: 10333397
• 负责人: Jeffrey C. Nolz
• 金额: $ 19.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-25 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10333397
• 关键词: Affinity; Aleuria aurantia lectin; Amino Acids; Antigens; Apoptosis; Asparagine; Binding; Biochemical; Biological; Bone Marrow; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Carbohydrates; Cell Adhesion; Cell physiology; Cells; Chimera organism; Chronic; Cytotoxic T-Lymphocytes; DNA; Data; Development; Enterobacteria phage P1 Cre recombinase; Enzymes; Fucose; Fucosyltransferase; Functional disorder; Generations; Genetic Code; Goals; Immunotherapy; In Vitro; Individual; Infection; Knockout Mice; Label; Lectin; Ligands; Link; Lymphocytic choriomeningitis virus; Mass Spectrum Analysis; Mediator of activation protein; Membrane Proteins; Modification; Molecular; Mus; Peptides; Physiology; Play; Polysaccharides; Post-Translational Protein Processing; Production; Proliferating; Proteins; RNA; Reagent; Role; Signal Transduction; Structure; T cell response; T-Cell Activation; T-Lymphocyte; Techniques; Testing; Tissues; Translating; Vaccine Design; Virus Diseases; anti-PD-L1; antigen-specific T cells; base; conditional knockout; cytokine; exhaust; exhaustion; functional restoration; gene product; glycosylation; glycosyltransferase; improved; in vivo; interleukin-21 receptor; mammalian genome; novel; preservation; protein degradation; protein function; receptor

PROJECT SUMMARY/ABSTRACT Post-translational carbohydrate modifications are important mediators of several cellular processes including protein turnover, cell adhesion, signal transduction, modulating receptor affinity for ligand, and apoptosis. However, in contrast to the well-established genetic code where biological information in DNA results in the generation of RNA which is then translated into protein, no such paradigm exists for predicting the vast array of possible post-translational glycosylation structures that could potentially decorate a given gene product. One such glycan modification has been defined as ‘core fucosylation’ of N-linked glycans, which occurs when L- fucose is covalently linked via an a1,6 linkage to the initial N-acetylglucosamine that originates from the asparagine amino acid. Although core fucosylation of N-linked glycans is necessary for normal development and physiology, whether these N-linked glycans are critical regulators of T cell activation and/or function is largely unknown. Here, we show that CD8+ T cells are decorated with N-linked glycans containing a core fucose and that the abundance of this specialized glycan modification increases significantly following their activation both in vitro and in vivo. Fucosyltransferase 8 (Fut8; a1,6-fucosyltransferase) is the only glycosyltransferase enzyme in the mammalian genome that can facilitate core fucosylation of N-linked glycans and we have now generated a novel Fut8 conditional knockout mouse that allows us to eliminate expression of Fut8 in a cell-specific manner. Using a T cell-specific cre-recombinase, our preliminary data show that expression of Fut8 and the subsequent generation of core fucosylated N-linked glycans is essential to maintain antigen-specific CD8+ T cell function (e.g. production of cytokines) during chronic viral infection. Here, we propose to use our new reagent to 1) identify the landscape of proteins expressed by activated CD8+ T cells that become decorated with core fucosylated N- linked glycans using a mass spectrometry approach and 2) to subsequently determine the biological relevance of this form of post-translational glycosylation in maintaining the function of antigen-specific CD8+ T cells during chronic viral infection.

项目总结/摘要 翻译后碳水化合物修饰是几种细胞过程的重要介质，包括 蛋白质周转、细胞粘附、信号转导、调节受体对配体的亲和力和凋亡。 然而，与DNA中的生物信息导致的 RNA的产生，然后被翻译成蛋白质，没有这样的范式存在预测的大量 可能的翻译后糖基化结构，可能潜在地修饰给定的基因产物。一 这种聚糖修饰被定义为N-连接聚糖的“核心岩藻糖基化”，当L- 岩藻糖通过α 1，6键共价连接至起始的N-乙酰葡糖胺， 天冬酰胺氨基酸。尽管N-连接聚糖的核心岩藻糖基化对于正常发育是必需的， 在生理学上，这些N-连接的聚糖是否是T细胞活化和/或功能的关键调节剂在很大程度上取决于它们是否是T细胞活化和/或功能的关键调节剂。 未知在这里，我们发现CD 8 + T细胞被含有核心岩藻糖的N-连接聚糖修饰， 这种专门的聚糖修饰的丰度在它们激活后显著增加， 在体外和体内。岩藻糖基转移酶8（Fut 8; α 1，6-岩藻糖基转移酶）是唯一的糖基转移酶 在哺乳动物基因组中，可以促进N-连接聚糖的核心岩藻糖基化， 一种新的Fut 8条件性敲除小鼠，使我们能够以细胞特异性方式消除Fut 8的表达。 使用T细胞特异性的cre重组酶，我们的初步数据显示Fut 8的表达和随后的细胞凋亡。 核心岩藻糖基化N-连接聚糖的产生对于维持抗原特异性CD 8 + T细胞功能至关重要 (e.g.细胞因子的产生）。在这里，我们建议使用我们的新试剂1）识别 由活化的CD 8 + T细胞表达的蛋白质的景观，这些细胞被核心岩藻糖基化的N- 使用质谱法测定连接的聚糖，以及2）随后测定生物相关性 这种形式的翻译后糖基化在维持抗原特异性CD 8 + T细胞的功能中的作用， 慢性病毒感染