Protein-Specific Polysialylation

聚唾液酸（PSA）是一种发育调节的抗粘附聚糖，被添加到神经细胞中， 粘附分子（NCAM），是五种哺乳动物多聚唾液酸化蛋白中最丰富的。的存在 NCAM N-聚糖上的PSA负性调节细胞粘附，并且对于多种重要的细胞粘附和/或细胞凋亡至关重要。 包括大脑发育，学习和记忆，神经元再生，以及生长和 癌细胞的侵袭性。我们的目标是了解蛋白质特异性的机制， 多聚唾液酸化以及多聚唾液酸转移酶（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（FN 1）是N-聚糖聚唾液酸化所必需的， 邻近的免疫球蛋白结构域（Ig 5）。我们已经解决了FN 1的晶体结构，并表明， 酸性表面补丁涉及polyST识别，并且是一个独特的螺旋，其连接链4和5 FN 1 β夹心结构的蛋白质，对于定位用于聚唾液酸化的Ig 5 N-聚糖至关重要。我们 提出实验来阐明蛋白质特异性多聚唾液酸化的机制，并验证这一假设 NCAM多聚唾液酸化需要聚ST-FN 1结合和Ig 5-FN 1相互作用。在目标I，我们将 使用聚唾液酸化的获得和丧失实验鉴定聚ST识别所需的FN 1残基。 在目的II中，我们将确定多聚ST-NCAM结合所需的FN 1序列和调节因子 使用免疫共沉淀和体外结合测定。在目标III中，我们将获得的晶体结构 Ig 5-FN 1，以确定这些结构域是否相互作用，FN 1螺旋在这种相互作用中的作用， 结合试验和旋转阴影电子显微镜，以评估替代的可能性， 聚ST允许在聚唾液酸化期间的瞬时Ig 5-FN 1相互作用。我们的长期目标是了解 聚唾液酸化的蛋白质特异性的基础，以便我们可以设计方法来消除或增强 发育和疾病期间的NCAM多聚唾液酸化。与公共卫生的相关性：聚唾液酸（PSA）是 抗粘附糖是专门添加到神经细胞粘附分子，NCAM。PSA对于 大脑发育、神经元再生并促进癌症侵袭。该项目的目标是 了解增加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多聚唾液酸化并调节其在发育和疾病期间对细胞粘附的作用。

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.