Mucin Site Specific O-Glycosylation

描述(申请人提供)：含有大量O-糖基化粘蛋白结构域的糖蛋白在保护细胞表面、调节细胞间相互作用、靶向蛋白质、调节炎症和免疫反应以及在肿瘤发生和转移中发挥重要的生物学作用。许多肿瘤抗原是粘蛋白样糖蛋白。O-糖基化结构域发挥着关键作用，这取决于它们的扩展结构和被一系列糖链结构修饰的能力。PpGalNAc转移酶家族将第一个糖a-GalNAc添加到多肽核心，而随后的转移酶通过向GalNAc添加糖来延长糖链。最近的研究表明，O-葡聚糖结构可以以一种依赖于肽序列的方式变化。这种O-糖基化调控的机制还不是很清楚，尽管肽序列和邻近基团的糖基化效应可能都很重要。这项工作的目的是详细地表征ppGalNAc转移酶家族以及向GalNAc添加糖(分别为β-Gal(1-3)、β-GIcNAc(1-3)和α-NeuNAc(2-6))的核心1、核心3和唾液酸基转移酶家族的肽底物特异性。在目标1中，将测定一系列ppGalNAc转移酶对几个高分子量脱附蛋白结构域和较小的随机多肽底物的位置特异性糖基化动力学。实验中的脱辅酶-粘蛋白糖基化动力学符合包含相邻糖基化和序列上下文效应的动力学模型。在目标2中，将研究在所有ppGalNAc转移酶中发现的蓖麻毒素样凝集素结构域的作用以及小分子竞争对手对ppGaINAc转移酶位点特异性糖基化的影响。在目标3中，添加到连接GalNAc的多肽的C3或C6位的转移酶的位置特异性糖基化动力学将被测定并如上所述地进行动力学模拟。通过这项工作，我们将对多肽序列和局部环境在调节O-葡聚糖生物合成起始步骤中的作用有了更好的理解。这将有助于更好地了解CD8、CD45、IgA1、PSGL-1和肿瘤粘蛋白抗原MUC1和CA125等生物重要分子的O-糖基化，这些分子的生物学特性取决于其O-糖基化状态。这些研究将产生合理预测特定部位O-糖链结构的基本工具。

DESCRIPTION (provided by applicant): Glycoproteins containing heavily O-glycosylated mucin domains play important biological roles protecting cell surfaces, modulating cell-cell interactions, targeting of proteins, regulating the inflammatory and immune responses, and in tumorogenisis and metastasis. A number of tumor antigens are mucin-like glycoproteins. O-glycosylated domains play critical roles that depend on their extended structures and ability to be decorated by an array of glycan structures. A family of ppGalNAc transferases adds the first sugar, a- GalNAc, to the peptide core while subsequent transferases elongate the glycan chain by adding sugars to GalNAc. Recent studies suggest that the O-glycan structures can vary in a peptide sequence dependent manner. The mechanisms behind this modulation of O-glycosylation is poorly understood, although both peptide sequence and neighboring group glycosylation effects may be important. It is the aim of this work to characterize in detail the peptide substrate specificities of the family of ppGalNAc transferases and the Core 1, Core 3 and sialyl transferases which add sugars (beta-Gal (1-3), beta-GIcNAc(1-3) and alpha-NeuNAc (2-6) respectively) to the GalNAc. In Aim 1 the site specific glycosylation kinetics of a series of ppGalNAc transferases against several high molecular weight apo-mucin domains and smaller random peptide substrates will be determined. Experimental apo-mucin glycosylation kinetics will be fit to a kinetic model incorporating neighboring glycosylation and sequence context effects. In Aim 2 the role of the ricin-like lectin domain found in all ppGalNAc transferases and the effects of small molecule competitors on ppGaINAc transferase site specific glycosylation will be investigated. In Aim 3 the site specific glycosylation kinetics of the transferases that add to the C3 or C6 positions of the peptide linked GalNAc will be determined and kinetically modeled as above. As a result of this work we will have obtained a sound understanding of the role of peptide sequence and local environment on the modulation of the initial steps of O-glycan biosynthesis. This will lead to a better understanding of the O-glycosylation of biologically important molecules such as CD8, CD45, IgA1, PSGL-1 and the tumor mucin antigens MUC1 and CA125, molecules whose biological properties are known to depend on their O-glycosylation state. Fundamental tools for the rational prediction of site specific O-glycan structures will result from these studies.