Protein-Specific Polysialylation

描述(申请人提供)：聚唾液酸(PSA)是一种发育调节、抗黏附的多糖，被添加到神经细胞黏附分子(NCAM)中，NCAM是五种哺乳动物聚唾液酸化蛋白质中含量最丰富的。NCAMN-糖链上PSA的存在负向调节细胞黏附，对脑发育、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都是至关重要的。我们在这个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基转移酶(Polysialyl Transfer ase，PolyST)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(FN1)是邻近免疫球蛋白结构域(Ig5)上发现的N-糖聚糖多聚糖所必需的。我们已经解决了FN1的晶体结构，并证明了酸性表面补丁参与了多ST识别，并且一个独特的螺旋连接了FN1β三明治结构的4和5链，对于定位Ig5 N-葡聚糖进行多唾液酸化至关重要。我们提出了一些实验来阐明蛋白质特异性多唾液酸化的机制，并验证了NCAM多唾液酸化需要PolyST-FN1结合和LG5-FN1相互作用的假设。在目标I中，我们将使用增益性和损失性聚唾液酸化实验来鉴定识别多聚ST所需的FN1残基。在AIM II中，我们将使用免疫共沉淀和体外结合分析来确定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，并使用结合分析和旋转阴影电子显微镜来评估另一种可能性，即在聚唾液分析过程中，PolySTs允许瞬时的LG5-FN1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，以便我们能够设计在发育和疾病过程中消除或增强NCAM多唾液酸化的方法。与公众健康相关：聚唾液酸(PSA)是一种抗粘连糖，专门添加到神经细胞黏附分子NCAM中。PSA对大脑发育、神经元再生至关重要，并促进癌症的侵袭性。本项目的目标是了解添加PSA的酶是如何识别NCAM的，以便我们能够设计出减少或增强NCAM多唾液酸化的方法，并调节其在发育和疾病过程中对细胞黏附的影响。

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.