Protein-Specific Polysialylation

蛋白质特异性多唾液酸化

基本信息

批准号：
7365348
负责人：
KAREN J. COLLEY
金额：
$ 8.72万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-07-01 至 2007-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7365348
关键词：
Adhesives Amino Acids Anabolism Attenuated Binding Biological Assay Birth Brain Catalytic Domain Cell Adhesion Charge Chimera organism Co-Immunoprecipitations Development Disease Docking Electron Microscopy Electrons Enzymes Fibronectins Goals Grant Growth Helix (Snails)Immunoglobulin Domain Immunoglobulins In Vitro Knockout Mice Learning Link Mediating Memory Modification Molecular Structure Natural regeneration Neural Cell Adhesion Molecules Neuraxis Neurons Polysaccharides Polysialic Acid Positioning Attribute Process Proteins Public Health Research Research Personnel Role Shadowing (Histology)Specificity Stretching Structure Surface Synaptic plasticity Testing Work X-Ray Crystallography axon guidance axon regeneration base cancer cell cancer invasiveness cell motility design ear helix polymerization programs protein protein interaction research study stem sugar

项目摘要

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 microscopyto 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.

聚唾液酸 (PSA) 是一种发育调节的抗粘附聚糖，添加到神经细胞中粘附分子（NCAM），五种哺乳动物多唾液酸化蛋白中最丰富的。的存在 NCAM N-聚糖上的 PSA 负向调节细胞粘附，对于多种重要的细胞粘附至关重要过程包括大脑发育、学习和记忆、神经元再生以及生长和发育癌细胞的侵袭力。我们本提案的目标是了解蛋白质特异性的机制聚唾液酸化以及聚唾液酸转移酶 (polyST) 如何识别 NCAM。我们已经证明了 NCAM 的第一个纤连蛋白 III 型重复序列 (FN1) 是 N-聚糖多唾液酸化所必需的相邻的免疫球蛋白结构域 (Ig5)。我们已经解析了 FN1 的晶体结构并表明酸性表面补丁涉及多ST识别，并且是一个独特的螺旋，它连接链4和5 FN1 β 夹心结构的结构对于定位 Ig5 N-聚糖进行多唾液酸化至关重要。我们提出实验来阐明蛋白质特异性多唾液酸化的机制并检验该假设 NCAM 聚唾液酸化需要 PolyST-FN1 结合和 lg5-FN1 相互作用。我们的目标是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，我们将确定 PolyST-NCAM 结合所需的 FN1 序列和调节因素使用免疫共沉淀和体外结合测定。在目标III中，我们将获得晶体结构 lg5-FN1 来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另一种可能性 PolyST 在多唾液酸化过程中允许短暂的 lg5-FN1 相互作用。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，以便我们可以设计消除或增强的方法发育和疾病过程中的 NCAM 多唾液酸化。与公共卫生的相关性：聚唾液酸 (PSA) 是专门添加到神经细胞粘附分子 NCAM 中的抗粘附糖。 PSA 至关重要大脑发育、神经元再生并促进癌症侵袭。该项目的目标是了解添加 PSA 的酶如何识别 NCAM，以便我们可以设计方法来减少或增强 NCAM 聚唾液酸化并调节其对发育和疾病期间细胞粘附的影响。