Protein-Specific Polysialylation

蛋白质特异性多唾液酸化

• 批准号: 7934437
• 负责人: KAREN J. COLLEY
• 金额: $ 21.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934437
• 关键词: Abbreviations; Adhesives; Affinity; Amino Acids; Anabolism; Binding; Biological Assay; Birth; Brain; Catalytic Domain; Cell Adhesion; Cell Adhesion Molecules; Charge; Chimera organism; Co-Immunoprecipitations; Cytoplasmic Tail; Development; Disease; Docking; Electron Microscopy; Endoglycosidase F; Enzymes; F-peptide; Fibronectins; Gel Chromatography; Goals; Growth; Immunoglobulin Domain; Immunoglobulins; In Vitro; Kinetics; Knockout Mice; Learning; Length; Link; Measures; Mediating; Membrane; Memory; Methods; Modification; Molecular Structure; Natural regeneration; Neural Cell Adhesion Molecules; Neuraxis; Neurons; Peptide N-glycohydrolase F; Polysaccharides; Polysialic Acid; Positioning Attribute; Process; Property; Proteins; Public Health; Research; Role; ST8Sia II; Shadowing (Histology); Specificity; Stretching; Structure; Surface; Surface Plasmon Resonance; Synaptic plasticity; Tail; Testing; Transmembrane Domain; Work; X-Ray Crystallography; axon guidance; axon regeneration; base; cancer cell; cancer invasiveness; cell motility; design; mutant; neuron development; polymerization; programs; protein protein interaction; research study; stem; sugar

DESCRIPTION (provided by applicant): Polysialic acid (PSA) is a developmental regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule (NCAM), the most abundant of five mammalian polysialylated proteins. The presence of PSA on NCAM N-glycans negatively modulates cell adhesion and is critical for a variety of important processes including brain development, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our goal in this proposal is to understand the mechanism of protein-specific polysialylation and how the polysialyltransferases (polySTs) recognize NCAM. We have demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of the N-glycans found on the adjacent immunoglobulin domain (Ig5). We have solved the crystal structure of FN1 and shown that an acidic surface patch is involved polyST recognition, and that a unique helix, which links the strands 4 and 5 of the FN1 beta sandwich structure, is critical for positioning the Ig5 N-glycans for polysialylation. We propose experiments to elucidate the mechanism of protein-specific polysialylation and to test the hypothesis that polyST-FN1 binding and an lg5-FN1 interaction are required for NCAM polysialylation. In aim I we will identifiy the FN1 residues required for polyST recognition using gain- and loss-of-polysialylation experiments. In aim II we will determine the FN1 sequences required for, and factors modulating, polyST-NCAM binding using co-immunoprecipitation and in vitro binding assays. In aim III, we will obtain the crystal structure of lg5-FN1 to determine whether these domains interact, the role of the FN1 helix in this interaction, and use binding assays and rotary shadowing electron microscopy to evaluate an alternate possibilty that the polySTs allow a transient lg5-FN1 interaction during polysialylation. Our long term goal is to understand the basis for the protein specificity of polysialylation so that we can design approaches to eliminate or enhance NCAM polysialylation during development and disease. Relevance to public health: Polysialic acid (PSA) is anti-adhesive sugar that is added specifically to the neural cell adhesion molecule, NCAM. PSA is critical for brain development, neuronal regeneration, and promotes cancer invasiveness. The goal of this project is to understand how the enzymes that add PSA recognize NCAM so that we can design approaches to diminish or enhance NCAM polysialylation and regulate its effects on cell adhesion during development and disease.

描述（由申请人提供）：聚唾液酸（PSA）是一种发育调节的抗粘附聚糖，添加到神经细胞粘附分子（NCAM）中，神经细胞粘附分子是五种哺乳动物多唾液酸化蛋白中最丰富的。 NCAM N-聚糖上存在的 PSA 会对细胞粘附产生负向调节作用，对于大脑发育、学习和记忆、神经元再生以及癌细胞的生长和侵袭等多种重要过程至关重要。我们在此提案中的目标是了解蛋白质特异性聚唾液酸化的机制以及聚唾液酸转移酶 (polyST) 如何识别 NCAM。我们已经证明，NCAM 的第一个纤连蛋白 III 型重复序列 (FN1) 是相邻免疫球蛋白结构域 (Ig5) 上发现的 N-聚糖的多唾液酸化所必需的。我们已经解析了 FN1 的晶体结构，并表明酸性表面斑块涉及 PolyST 识别，并且连接 FN1 β 夹心结构的 4 链和 5 链的独特螺旋对于定位 Ig5 N-聚糖进行聚唾液酸化至关重要。我们提出实验来阐明蛋白质特异性聚唾液酸化的机制，并测试 NCAM 聚唾液酸化需要 PolyST-FN1 结合和 lg5-FN1 相互作用的假设。在目标 I 中，我们将使用多唾液酸化的获得和损失实验来鉴定 PolyST 识别所需的 FN1 残基。在目标 II 中，我们将使用免疫共沉淀和体外结合测定来确定 PolyST-NCAM 结合所需的 FN1 序列和调节因素。在目标 III 中，我们将获得 lg5-FN1 的晶体结构，以确定这些结构域是否相互作用、FN1 螺旋在这种相互作用中的作用，并使用结合测定和旋转阴影电子显微镜来评估 PolyST 在聚唾液酸化过程中允许瞬时 lg5-FN1 相互作用的另一种可能性。我们的长期目标是了解多唾液酸化的蛋白质特异性的基础，以便我们能够设计在发育和疾病期间消除或增强 NCAM 多唾液酸化的方法。与公共健康的相关性：聚唾液酸 (PSA) 是一种抗粘附糖，专门添加到神经细胞粘附分子 NCAM 中。 PSA 对于大脑发育、神经元再生和促进癌症侵袭至关重要。该项目的目标是了解添加 PSA 的酶如何识别 NCAM，以便我们能够设计减少或增强 NCAM 聚唾液酸化的方法，并调节其对发育和疾病期间细胞粘附的影响。