Mucin Site Specific O-Glycosylation

粘蛋白位点特异性 O-糖基化

• 批准号: 7013227
• 负责人: THOMAS A GERKEN
• 金额: $ 26.89万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-05 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7013227
• 关键词: N acetylgalactosamine; active sites; carbohydrate sequence; carbohydrate structure; cell line; cytoprotection; enzyme substrate; galactosyltransferases; glycosylation; isozymes; matrix assisted laser desorption ionization; mucins; neoplastic cell; nuclear magnetic resonance spectroscopy; recombinant proteins; transferase

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.

描述（由申请人提供）：含有重度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聚糖结构修饰的能力。 ppGalNAc 转移酶家族将第一个糖（a-GalNAc）添加到肽核心，而随后的转移酶通过将糖添加到 GalNAc 来延长聚糖链。最近的研究表明，O-聚糖结构可以以肽序列依赖性方式变化。尽管肽序列和邻近基团糖基化效应可能很重要，但这种 O-糖基化调节背后的机制知之甚少。本工作的目的是详细表征 ppGalNAc 转移酶家族以及向 GalNAc 添加糖（分别为 β-Gal (1-3)、β-GlcNAc(1-3) 和 α-NeuNAc (2-6)）的 Core 1、Core 3 和唾液酸转移酶的肽底物特异性。在目标 1 中，将确定一系列 ppGalNAc 转移酶针对几个高分子量 apo-粘蛋白结构域和较小的随机肽底物的位点特异性糖基化动力学。实验性脱辅基粘蛋白糖基化动力学将适合包含邻近糖基化和序列背景效应的动力学模型。在目标 2 中，将研究所有 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剂对 ppGalNAc 转移酶位点特异性糖基化的影响。在目标 3 中，将确定添加至肽连接的 GalNAc 的 C3 或 C6 位置的转移酶的位点特异性糖基化动力学，并按上述方法进行动力学建模。通过这项工作，我们将对肽序列和局部环境在 O-聚糖生物合成初始步骤的调节中的作用有一个深入的了解。这将有助于更好地理解生物学重要分子的 O-糖基化，例如 CD8、CD45、IgA1、PSGL-1 以及肿瘤粘蛋白抗原 MUC1 和 CA125，这些分子的生物学特性取决于其 O-糖基化状态。这些研究将产生用于合理预测位点特异性 O-聚糖结构的基本工具。