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.

项目摘要/摘要 T细胞生物学的几乎每一个方面都在一定程度上由多种T细胞的相对表达决定。 细胞表面蛋白。例如，共刺激和共抑制受体的相对表达影响 支持与抗炎的结果。N-糖基化是一种关键但知之甚少的细胞调节因子- 表面蛋白周转。几乎每个细胞表面和分泌的蛋白质都被共价连接的蛋白质修饰。 天冬酰胺(N)残基上的复合碳水化合物。这些N-糖链由大量的 沿着分泌途径的糖基化酶，产生许多可能的糖链结构。N-葡聚糖 分支途径是相对简单的高甘露糖结构和更多 携带许多碳水化合物结合家族识别的糖表位的复杂N-糖聚糖 蛋白质，称为凝集素。分支途径控制N-乙酰乳糖胺(LacNAc)的掺入 N-葡聚糖，由半乳糖凝集素家族的可溶性凝集素识别。在细胞表面，相互作用 带有携带LacNAc单位的糖蛋白的多价可溶Galectins导致分子的形成 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，受体在 膜平面和内吞速率。部分是通过调节细胞表面CD4，CD8， IL-2Rα、CTLA-4和T细胞受体，N-糖链分支调节T细胞发育 信号、T细胞激活、T细胞增殖和促炎分化与抗炎分化。 基于对这些受体的研究，出现了一个晶格模型，它是细胞的单向调节器-- 表面滞留，防止糖蛋白丢失，促进细胞表面滞留。然而，反- 最近出现的这种模型的例子表明，一些受体在 完全相反的方式。因此，需要对分支监管变化进行全面和公正的分析。 此外，对促进某些受体表达的机制的理解 也缺乏对他人表达的阻碍。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。我们已经开发出一种方法来 在蛋白质组水平上研究分支对细胞表面表达的影响。我们提供证明- 这种方法是可靠和信息丰富的，并建议使用它来解决以下突出方面 T细胞生物学。在Aim1中，我们将把这种方法扩展到更具生理相关性的初级T细胞和 研究一系列N-葡聚糖的分支状态，以得出一个完整和信息丰富的图景。我们也会适应 我们的方法是在全球范围内确定分支调节蛋白的内吞作用和循环率。在目标2中， 我们将检验这一假说，即分子筛蛋白介导的内吞作用途径中的货物适配蛋白相互配合。 通过N-糖链的分支来差异化地调节受体的周转和表达。这些研究将结合在一起 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。