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，糖基化的生理意义可以通过许多实例来证明，其中可变的糖基化损害蛋白质功能并导致发育缺陷和疾病。尽管如此，控制哪些聚糖在蛋白质上组装的因素还不是很清楚。多唾液化是蛋白质特异性修饰的一个显著例子，它可以显著改变蛋白质的功能。聚唾液酸（polySia）以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、突触可塑性和神经系统发育中的作用而闻名。在受损的周围神经元上，PolySia也被上调，促进它们的再生，在几种不同类型的癌症表面，PolySia促进它们的生长和侵袭性。值得注意的是，除了修饰自己的n -聚糖的多唾液基转移酶（polySTs）外，只在五种蛋白质上发现了polySia。最近发现的两种polyST底物，突触粘附分子SynCAM 1和信号蛋白和VEGF共受体neuropilin-2 (NRP-2)，表明polySia的作用可能比以前认为的更广泛，并提出了polyST如何识别和修饰这些不同底物的问题。我们的长期目标是确定蛋白质特异性聚唾液化的机制，哪些因素调节聚sia链在特定底物上的聚合，以及聚sia如何调节其修饰的蛋白质的功能。在这个提议中，我们将测试这样一个假设，即聚合酶链能识别其底物的共同氨基酸和结构特征，这种相互作用允许在底物的聚糖上聚合聚sia链，然后是聚合酶链相互作用，促进链的进一步延伸。为了做到这一点，我们将评估NCAM、SynCAM 1和NRP-2的多碱基识别和多唾液化的结构域和序列要求，并确定保守的多碱基区域中的残基是否介导底物蛋白和/或多sia链相互作用，以促进蛋白质特异性多sia链聚合。我们还将验证一个假设，即SynCAM 1和NRP-2的茎区长度的变化（由选择性剪接产生）改变了它们与膜相关的聚合体的排列，并以细胞和组织特异性的方式控制这些蛋白的聚唾液化。我们预计这些研究将使我们能够确定多唾液酰化过程中受生理调节的点，并且这些点将适用于实验和治疗操作，以控制发育，修复和疾病期间的底物多唾液酰化和功能。

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.