Mechanism and Regulation of Protein-Specific Polysialylation

描述（由申请人提供）：超过一半的人类蛋白质是糖基化的，糖基化的生理学意义通过多种实例来举例说明，其中可变糖基化损害蛋白质功能并导致发育缺陷和疾病。尽管如此，控制哪些聚糖组装在蛋白质上的因素还不清楚。聚唾液酸化是蛋白质特异性修饰的一个突出例子，可以显着改变蛋白质功能。聚唾液酸（polySia）最为人所知的是其阻断神经细胞粘附分子（NCAM）依赖性细胞粘附和信号传导的能力，以及其在细胞迁移、轴突引导、突触可塑性、神经系统发育中的作用。PolySia也在受损的外周神经元上上调，并促进其再生，以及在几种不同类型的癌症表面上，它促进其生长和侵袭性。值得注意的是，除了修饰其自身N-聚糖的聚唾液酸转移酶（polyST）之外，仅在五种蛋白质上发现polySia。最近鉴定的两个polyST底物，SynCAM 1，突触粘附分子，和神经纤毛蛋白-2（NRP-2），脑信号蛋白和VEGF共受体，表明polySia的作用可能比以前认为的更广泛，并提出了问题的polyST如何识别和修改这些不同的底物。我们的长期目标是确定蛋白质特异性聚唾液酸化的机制，什么因素调节polySia链在特定底物上的聚合，以及polySia如何调节其修饰的蛋白质的功能。在本提案中，我们将检验以下假设：polyST识别其底物的共同氨基酸和结构特征，并且这种相互作用允许polySia链在底物聚糖上的初始聚合，并且随后是促进进一步链延伸的polyST-polySia链相互作用。为此，我们将评估NCAM、SynCAM 1和NRP-2的polyST识别和多聚唾液酸化的结构域和序列要求，并确定保守的polyST多碱基区中的残基是否介导底物蛋白和/或polySia链相互作用以促进蛋白特异性polySia链聚合。我们还将检验以下假设：通过选择性剪接产生的SynCAM 1和NRP-2的茎区长度的变化改变了它们与膜相关polyST的对齐，并以细胞和组织特异性方式控制这些蛋白质的聚唾液酸化。我们预计，这些研究将使我们能够确定在聚唾液酸化过程中的点，受到生理调节，并将B服从实验和治疗操作，以控制底物聚唾液酸化和功能在发展，修复和疾病。 公共卫生相关性：聚唾液酸是一种发育调节糖聚合物，存在于一小群粘附和信号蛋白上。它对神经系统发育至关重要，并在细胞迁移、突触可塑性、神经元再生以及各种癌症的生长和侵袭中发挥作用。拟议的工作将阐明聚唾液酸化的机制，并确定生理调节点，可以通过实验和治疗来控制聚唾液酸化蛋白在发育和疾病过程中的功能。

DESCRIPTION (provided by applicant): More than half of all human proteins are glycosylated, and the physiological significance of glycosylation is exemplified by the numerous instances in which variable glycosylation compromises protein function and causes developmental defects and disease. Despite this, the factors that control which glycans are assembled on proteins are not well understood. Polysialylation is a striking example of a protein specific modification that can dramatically change protein function. Polysialic acid (polySia) is best known for its ability to block neural cell adhesion molecule (NCAM)-dependent cell adhesion and signaling, and for its roles in cell migration, axon guidance, synaptic plasticity, an nervous system development. PolySia is also upregulated on damaged peripheral neurons and facilitates their regeneration, and on the surface of several different types of cancers where it promotes their growth and invasiveness. Remarkably, polySia is found on only five proteins in addition to the polysialyltransferases (polySTs) that modify their own N-glycans. The recent identification of two of these polyST substrates, SynCAM 1, a synaptic adhesion molecule, and neuropilin-2 (NRP-2), a semaphorin and VEGF co-receptor, suggests that the roles of polySia may be more extensive than previously thought, and raises the question of how the polySTs recognize and modify these distinct substrates. Our long-term objectives are to determine the mechanism of protein specific polysialylation, what factors regulate the polymerization of polySia chains on specific substrates, and how polySia modulates the functions of the proteins it modifies. In this proposal we will test the hypothesis that the polySTs recognize common amino acid and structural features of their substrates and that this interaction allows an initial polymerization of the polySia chain on a substrate's glycans, and that this is followed by a polyST-polySia chain interaction that promotes further chain elongation. To do this we will evaluate the domain and sequence requirements for polyST recognition and polysialylation of NCAM, SynCAM 1 and NRP-2, and determine whether residues in a conserved polyST polybasic region mediate substrate protein and/or polySia chain interaction to promote protein specific polySia chain polymerization. We will also test the hypothesis that changes in the length of the stalk regions of SynCAM 1 and NRP-2, generated by alternative splicing, alter their alignment with membrane- associated polySTs and control the polysialylation of these proteins in a cell- and tissue-specific manner. We anticipate that these studies will allow us to identify points in the polysialylation process that are subject to physiological regulation, and that will b amenable to experimental and therapeutic manipulations to control substrate polysialylation and function during development, repair, and disease. PUBLIC HEALTH RELEVANCE: Polysialic acid is a developmentally regulated sugar polymer found on a small group of adhesive and signaling proteins. It is critical for nervous system development, and plays roles in cell migration, synaptic plasticity, neuronal regeneration, and the growth and invasiveness of a wide variety of cancers. The proposed work will elucidate the mechanism of polysialylation, and identify points of physiological regulation that can be experimentally and therapeutically manipulated to control the functions of polysialylated proteins during development and disease.