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: 10646138
• 负责人: Haik Mkhikian
• 金额: $ 46.71万
• 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/10646138
• 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; Protein Secretion; 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; carbohydrate binding protein; carbohydrate receptor; 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.

项目总结/摘要 T细胞生物学的几乎每一个方面都部分地由大量T细胞的相对表达决定。 细胞表面蛋白例如，共刺激受体和共抑制受体的相对表达影响了细胞的增殖。 促炎和抗炎的结果。N-糖基化是一个关键的，但了解甚少的调节细胞， 表面蛋白周转事实上，每一个细胞表面和分泌的蛋白质都被共价连接的 天冬酰胺（N）残基处的复合碳水化合物。这些N-聚糖被大量的 糖基化酶沿着分泌途径，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的甘露糖结构之间的关键通道。 复杂的N-聚糖，其携带被许多碳水化合物结合蛋白家族识别的糖表位 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，其被可溶性凝集素的半乳糖凝集素家族识别。在细胞表面， 具有携带LacNAc单元的糖蛋白的多价可溶性半乳糖凝集素，导致形成分子 格子状结构。这种半乳糖凝集素-糖蛋白晶格影响细胞表面的组织结构， 膜平面和内吞率。部分通过调节细胞表面CD 4，CD 8， IL 2 R α、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细胞活化、T细胞增殖和促炎与抗炎分化。 基于对这些受体的研究，出现了一种模型，即晶格是细胞的单向调节器， 阻止糖蛋白损失并促进细胞表面保留的表面保留。但是，反- 最近出现了这种模型的例子，证明一些受体在细胞中受到调节， 相反的方式。因此，一个全面的和公正的分析分支调节的变化是必要的。 此外，对促进某些受体表达的机制的理解， 也不妨碍他人的表达。如果没有如此详细的信息， 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， 在蛋白质组水平上检查分支介导的对细胞表面表达的影响。我们提供证据- 我认为这种方法是可靠的和信息丰富的概念，并建议用它来解决这一突出的方面， T细胞生物学在Aim 1中，我们将把这种方法扩展到生理学上更相关的原代T细胞， 检查一系列N-聚糖分支状态，以获得完整的信息图片。我们也会适应 我们的方法，以全球确定内吞和回收率的分支调节蛋白质。在目标2中， 我们将检验网格蛋白介导的内吞途径中的货物衔接蛋白协同作用的假设 具有N-聚糖分支以差异调节受体周转和表达。这些研究将 极大地增加了我们对分支介导的T细胞生物学调节的知识。