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，糖基化的生理意义通过大量可变糖基化损害蛋白质功能并导致发育缺陷和疾病的实例来例证。尽管如此，控制哪些糖链在蛋白质上组装的因素还不是很清楚。聚唾液酸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(Polysialic acid，PolySia)具有阻断神经细胞黏附分子(NCAM)依赖的细胞黏附和信号转导的能力，在细胞迁移、轴突引导、突触可塑性、神经系统发育等方面发挥重要作用。PolySia还上调受损的外周神经元，促进其再生，并在几种不同类型的癌症表面促进其生长和侵袭性。值得注意的是，除了修饰自己N-糖链的聚唾液酸转移酶(Polysialyl Transfer ase，PolyST)外，只有五种蛋白质上发现了PolySia。最近对突触黏附分子SynCAM1和信号素和血管内皮生长因子共同受体NRP-2这两种PolyST底物的发现表明，PolySia的作用可能比先前认为的更广泛，并提出了PolyST如何识别和修饰这些不同的底物的问题。我们的长期目标是确定蛋白质特异性聚唾液酸化的机制，哪些因素调节特定底物上PolySIA链的聚合，以及PolySia如何调节其修饰的蛋白质的功能。在这个提议中，我们将检验这样的假设，即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底物的多糖上的PolySia链的初始聚合，并且随后是PolyST-PolySia链相互作用，促进进一步的链延长。为此，我们将评估NCAM、SynCAM 1和NRP-2的多聚ST识别和多聚唾液酸化的结构域和序列要求，并确定保守的多聚ST多碱区的残基是否介导底物蛋白质和/或多聚Sia链相互作用，以促进蛋白质特异的多聚SIA链聚合。我们还将检验这样一种假设，即由选择性剪接产生的SynCAM 1和NRP-2茎区域长度的变化，改变了它们与膜相关多聚ST的排列，并以细胞和组织特异性的方式控制这些蛋白质的多唾液酸化。我们预计，这些研究将使我们能够确定聚唾液酸化过程中受生理调节的点，并将适合于实验和治疗操作来控制底物聚唾液酸化及其在发育、修复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.