NMR of cell surface oligosaccharides

细胞表面寡糖的 NMR

• 批准号: 8063027
• 负责人: JAMES H. PRESTEGARD
• 金额: $ 36.39万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063027
• 关键词: Anisotropy; Autoimmune Diseases; B-Cell Activation; Binding; C-Type Lectins; CD22 antigen; Carbohydrates; Cell Communication; Cell surface; Cells; Characteristics; Chemicals; Collection; Communication; Complex; Data; Data Collection; Dendritic Cells; Detection; Development; Environment; Fucose; Galactose; Galactose Binding Lectin; Glycoproteins; Grant; Health; Human Biology; Immune; Immune response; Indium; Inflammation; Investigation; Isotopes; Label; Lead; Lectin; Link; Lymphocyte antigen CD50; Mass Spectrum Analysis; Mediating; Membrane; Membrane Glycoproteins; Methodology; Methods; NMR Spectroscopy; Nature; Nuclear Magnetic Resonance; Oligosaccharides; Pathway interactions; Polysaccharides; Preparation; Procedures; Protein-Carbohydrate Interaction; Proteins; Regulation; Research Personnel; Residual state; Scheme; Sialic Acids; Signal Transduction; Site; Structure; Surface; System; T cell response; Work; base; drug development; glycosylation; granulocyte; human SIGLEC5 protein; human disease; improved; intermolecular interaction; neutrophil; pathogen; protein expression; protein protein interaction; receptor; response; sialic acid-binding lectin

DESCRIPTION (provided by applicant): Cell-cell interactions are often mediated by the recognition of N- or O-linked glycans on cell- surface glycoproteins by endogenous lectins. These lectins are often themselves glycoproteins, anchored to cells by trans-membrane segments that generate intra-cellular signals. Glycan mediated interaction of multiple lectin molecules can, therefore, be an important element in regulation of cell to cell signaling. For the past several years this grant has supported the development of nuclear magnetic resonance (NMR) methods for the investigation of structural aspects of the interaction of isolated oligosaccharides with soluble lectins and applied them primarily to a subset of lectins that binds galactose terminated oligosaccharides. We now propose to extend these studies to lectins targeting sialic acid and fucose containing oligosaccharides, and to develop methods that will allow these investigations to proceed when the oligosaccharides are glycans covalently linked to glycoproteins. Modulation of immune response by specific cell-cell interactions is a good example where glycan mediated protein-protein interaction is an issue. B-cell activation is regulated by cis- versus trans- interaction of siglecs (sialic acid binding lectins) on the surface of immune cells. DC-SIGN (dendritic cell -specific ICAM-3 grabbing nonintegrin), a C-type lectin that binds fucosylated oligosaccharides and interacts with ICAM-3 (intercellular adhesion molecule-3) also is believed to modulate an immune response. The proposed studies will provide a structural basis for understanding of these important regulatory mechanisms. The studies will also lead to the development of new methodology that can be applied to a variety of other problems in human biology where glycans mediate protein-protein interactions. The specific aims include expression of siglec domain constructs suitable for NMR studies in glycosylated and non-glycosylated forms, remodeling of glycan residues on siglecs to improve glycan homogeneity and add isotopically labeled sialic acids, development of new NMR resonance assignment strategies for these labeled glycans, collection and interpretation of NMR data to define structure and dynamics of these glycans, and development of parallel strategies that would allow structural characterization of fucose containing glycans important to function of the DC-SIGN system. PUBLIC HEALTH RELEVANCE: The interaction of cell-surface oligosaccharides with protein receptors is a primary means by which cells sense their environment. These can be beneficial in cases of detection and response to pathogens; it can be detrimental in aberrant response in autoimmune disease and inflammation. A structural understanding of the nature of these interactions is an important basis for the development of drugs that can modulate these responses. This project attempts to define the structural and dynamic characteristics of carbohydrate-protein interactions, not just with isolated carbohydrates, but with carbohydrates attached to the glycoproteins that are parts of communication pathways. New NMR methods for working with carbohydrates on glycoproteins are developed and applied to glycan-mediated interactions in systems important to immune response and human disease.

描述（由申请人提供）：细胞 - 细胞相互作用通常是通过内源性凝集素在细胞表面糖蛋白上识别N-或O连接的聚糖的介导的。这些凝集素通常是糖蛋白，通过产生细胞内信号的反膜段锚定在细胞上。因此，多种凝集素分子的聚糖介导的相互作用可以成为调节细胞至细胞信号传导的重要元素。在过去的几年中，该赠款支持了核磁共振（NMR）方法的发展，用于研究分离的寡糖与可溶性凝集素的相互作用的结构方面，并主要将其应用于结合半乳糖终止寡糖的凝集素的子集。现在，我们建议将这些研究扩展到靶向唾液酸和含有寡糖的岩藻糖的凝集素，并开发方法，这些方法将使这些研究允许在寡糖与糖蛋白共价链接时进行这些研究。特定细胞 - 细胞相互作用对免疫反应的调节是一个很好的例子，其中聚糖介导的蛋白质 - 蛋白质相互作用是一个问题。 B细胞激活受免疫细胞表面上sigLec（唾液酸结合凝集素）的顺式与转换的调节。 DC-SIGN（树突状细胞特异性ICAM-3抓取非整合蛋白），一种结合葡萄糖基化的寡糖并与ICAM-3（细胞间粘附分子-3）相互作用的C型凝集素也被认为可以调节免疫反应。拟议的研究将为理解这些重要的调节机制提供结构性基础。这些研究还将导致新方法的发展，这些方法可以应用于聚糖介导蛋白质蛋白质相互作用的人类生物学中的其他各种问题。 The specific aims include expression of siglec domain constructs suitable for NMR studies in glycosylated and non-glycosylated forms, remodeling of glycan residues on siglecs to improve glycan homogeneity and add isotopically labeled sialic acids, development of new NMR resonance assignment strategies for these labeled glycans, collection and interpretation of NMR data to define structure and dynamics of这些聚糖以及平行策略的发展，这些策略将允许对含聚糖的结构表征对DC-SIGN系统的功能很重要。公共卫生相关性：细胞表面寡糖与蛋白质受体的相互作用是细胞感知其环境的主要手段。在检测和对病原体的反应的情况下，这些可能是有益的。在自身免疫性疾病和炎症中的异常反应可能是有害的。对这些相互作用性质的结构理解是开发可以调节这些反应的药物的重要基础。该项目试图定义碳水化合物 - 蛋​​白质相互作用的结构和动态特征，不仅与孤立的碳水化合物，而且还与碳水化合物连接到糖蛋白上，这是通信途径的一部分。开发了与糖蛋白上碳水化合物一起使用的新NMR方法，并应用于对免疫反应和人类疾病重要的系统中的聚糖介导的相互作用。