Protein-Specific Polysialylation

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，被添加到神经细胞粘附分子（NCAM）中，NCAM是五种哺乳动物聚唾液酸化蛋白中含量最多的。PSA在NCAM n -聚糖上的存在可以负向调节细胞粘附，并且对多种重要过程至关重要，包括大脑发育、学习和记忆、神经元再生以及癌细胞的生长和侵袭。我们的目标是了解蛋白质特异性多唾液酰化的机制以及多唾液酰转移酶（polySTs）如何识别NCAM。我们已经证明，NCAM的第一个纤维连接蛋白III型重复序列（FN1）是邻近免疫球蛋白结构域（Ig5）上n -聚糖的多唾液化所必需的。我们已经解决了FN1的晶体结构，并表明酸性表面斑块参与聚st识别，并且连接FN1 β三明治结构的第4股和第5股的独特螺旋对于定位Ig5 n -聚糖进行聚唾液化至关重要。我们提出实验来阐明蛋白质特异性多唾液化的机制，并验证polyST-FN1结合和lg5-FN1相互作用是NCAM多唾液化所必需的假设。在目标1中，我们将使用聚唾液化的增益和损失实验确定聚st识别所需的FN1残基。在目的II中，我们将使用共免疫沉淀和体外结合试验确定polyST-NCAM结合所需的FN1序列和调节因子。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在多聚化过程中聚合物允许短暂的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化蛋白特异性的基础，这样我们就可以设计出在发育和疾病期间消除或增强NCAM多唾液化的方法。与公共卫生相关：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，以便我们可以设计减少或增强NCAM多唾液化的方法，并调节其在发育和疾病期间对细胞粘附的影响。

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.