Differential regulation of T cell plasma membrane proteins by N-glycan branching and clathrin-mediated endocytosis

N-聚糖分支和网格蛋白介导的内吞作用对 T 细胞质膜蛋白的差异调节

• 批准号: 10295564
• 负责人: Haik Mkhikian
• 金额: $ 47.1万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10295564
• 关键词: Adaptor Signaling Protein; Affect; Anti-Inflammatory Agents; Asparagine; CD4 Positive T Lymphocytes; CD8B1 gene; CTLA4 gene; Carbohydrates; Cell Surface Proteins; Cell Surface Receptors; Cell membrane; Cell physiology; Cell surface; Cellular biology; Clathrin; Complex; Coupled; Data; Diagnostic; Endocytosis; Endocytosis Pathway; Endoplasmic Reticulum; Enzymes; Epitopes; Face; Family; Galactose Binding Lectin; Glycoproteins; Goals; Immunity; Individual; Inflammation; Inflammatory; Knowledge; Lectin; Letters; Mannose; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Proteins; Mentors; Methodology; Methods; Modeling; Molecular; Monosaccharides; Mutagenesis; N-acetyllactosamine; Outcome; Pathway interactions; Physiological; Polysaccharides; Proteins; Proteome; Proteomics; Receptor Signaling; Recycling; Regulation; Rest; Role; Structure; Surface; T cell regulation; T-Cell Activation; T-Cell Development; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; base; carbohydrate binding protein; crosslink; extracellular; glycosylation; immunoregulation; insight; knowledgebase; novel strategies; protein degradation; protein expression; receptor; receptor expression; response; therapeutic target; therapeutically effective; virtual

PROJECT SUMMARY/ABSTRACT Nearly every aspect of T cell biology is determined in part by the relative expression of a multitude of cell-surface proteins. For example, the relative expression of co-stimulatory and co-inhibitory receptors impact pro- versus anti-inflammatory outcomes. N-glycosylation is a critical but poorly understood regulator of cell- surface protein turnover. Virtually every cell-surface and secreted protein is modified with covalently attached complex carbohydrates at asparagine (N) residues. These N-glycans are variably edited by a large number of glycosylation enzymes along the secretory pathway, producing many possible glycan structures. The N-glycan branching pathway serves as a key gateway between relatively simple high-mannose structures and more complex N-glycans that carry glyco-epitopes recognized by numerous families of carbohydrate-binding proteins, known as lectins. The branching pathway controls N-acetyllactosamine (LacNAc) incorporation into N-glycans, which are recognized by the galectin family of soluble lectins. At the cell surface, the interaction of multivalent soluble galectins with glycoproteins carrying LacNAc units, leads to the formation of a molecular lattice-like structure. This galectin-glycoprotein lattice affects cell surface organization, receptor mobility in the plane of the membrane, and endocytosis rates. In part by regulating cell surface expression of CD4, CD8, IL2Rα, CTLA-4, and the T cell receptor (TCR), N-glycan branching regulates T cell development, TCR signaling, T cell activation, T cell proliferation and pro-inflammatory versus anti-inflammatory differentiation. Based on studies of these receptors, a model has emerged of the lattice as a unidirectional regulator of cell- surface retention which opposes glycoprotein loss and promotes cell surface retention. However counter- examples to this model have recently emerged demonstrating that some receptors are regulated in the opposite manner. Thus a comprehensive and unbiased analysis of branching regulated changes is needed. Furthermore, an understanding of the mechanisms involved that promote expression of some receptors while hindering expression of others is also lacking. Without such detailed information, therapeutic targeting of the complex glycosylation pathways that regulate T cell function will be limited. We have developed an approach to examine branching mediated effects on cell-surface expression at the proteome scale. We provide proof-of- concept that this approach is reliable and informative and propose to use it to tackle this outstanding aspect of T cell biology. In Aim1, we will extend this approach to more physiologically relevant primary T cells and examine a range of N-glycan branching states to derive a complete and informative picture. We will also adapt our approach to globally determine endocytosis and recycling rates of branching regulated proteins. In Aim 2, we will test the hypothesis that cargo adapter proteins in the clathrin-mediated endocytosis pathway cooperate with N-glycan branching to differentially regulate receptor turnover and expression. Together these studies will dramatically increase our knowledge of branching mediated regulation of T cell biology.

项目概要/摘要 T 细胞生物学的几乎每个方面都部分取决于多种细胞的相对表达 细胞表面蛋白质。例如，共刺激和共抑制受体的相对表达影响 促炎结果与抗炎结果。 N-糖基化是细胞的关键但人们知之甚少的调节因子 表面蛋白质周转。事实上，每个细胞表面和分泌的蛋白质都经过共价连接修饰 天冬酰胺 (N) 残基上的复合碳水化合物。这些 N-聚糖经过大量不同的编辑 沿着分泌途径的糖基化酶，产生许多可能的聚糖结构。 N-聚糖 分支途径是相对简单的高甘露糖结构和更多结构之间的关键门户 复杂的 N-聚糖，携带被众多碳水化合物结合家族识别的糖表位 蛋白质，称为凝集素。分支途径控制 N-乙酰基乳糖胺 (LacNAc) 掺入 N-聚糖，被可溶性凝集素的半乳糖凝集素家族识别。在细胞表面，相互作用 多价可溶性半乳糖凝集素与携带 LacNAc 单位的糖蛋白，导致分子的形成 格状结构。这种半乳糖凝集素-糖蛋白晶格影响细胞表面组织、受体的迁移性 膜平面和内吞速率。部分通过调节细胞表面 CD4、CD8 的表达， IL2Rα、CTLA-4 和 T 细胞受体 (TCR)、N-聚糖分支调节 T 细胞发育、TCR 信号传导、T 细胞激活、T 细胞增殖以及促炎与抗炎分化。 基于对这些受体的研究，出现了一个晶格模型作为细胞的单向调节器。 表面保留可防止糖蛋白损失并促进细胞表面保留。然而反 最近出现的该模型的例子表明，某些受体在 相反的方式。因此，需要对分支调节变化进行全面且公正的分析。 此外，了解促进某些受体表达的机制 也缺乏阻碍他人表达的行为。如果没有这样详细的信息，治疗靶向 调节 T 细胞功能的复杂糖基化途径将受到限制。我们开发了一种方法 在蛋白质组规模上检查分支介导对细胞表面表达的影响。我们提供证明- 认为这种方法可靠且信息丰富，并建议用它来解决这一突出问题 T 细胞生物学。在 Aim1 中，我们将把这种方法扩展到生理上更相关的原代 T 细胞和 检查一系列 N-聚糖分支状态以获得完整且信息丰富的图片。我们也会适应 我们在全球范围内确定分支调节蛋白的内吞作用和回收率的方法。在目标 2 中， 我们将测试网格蛋白介导的内吞作用途径中的货物衔接蛋白合作的假设 具有 N-聚糖分支来差异调节受体周转和表达。这些研究将共同 极大地增加了我们对 T 细胞生物学分支介导调节的了解。