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）：含有高度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-细胞相互作用、靶向蛋白、调节炎症和免疫应答以及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-糖基化结构域发挥关键作用，这取决于它们的延伸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α- 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。这种O-糖基化调节背后的机制知之甚少，尽管肽序列和相邻基团糖基化效应可能都很重要。本工作的目的是详细表征ppGalNAc转移酶家族和核心1、核心3和唾液酸转移酶的肽底物特异性，所述核心1、核心3和唾液酸转移酶将糖（分别为β-Gal（1-3）、β-GlcNAc（1-3）和α-NeuNAc（2-6））添加到GalNAc。在目的1中，将确定一系列ppGalNAc转移酶针对几种高分子量脱辅基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。实验脱辅基粘蛋白糖基化动力学将拟合到并入相邻糖基化和序列背景效应的动力学模型。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者对ppGalNAc转移酶位点特异性糖基化的影响。在目的3中，将测定添加到肽连接的GalNAc的C3或C6位置的转移酶的位点特异性糖基化动力学，并如上所述进行动力学建模。作为这项工作的结果，我们将获得一个良好的理解的作用肽序列和局部环境的调制的初始步骤的O-聚糖生物合成。这将导致更好地理解生物学上重要分子的O-糖基化，例如CD 8、CD 45、IgA 1、PSGL-1和肿瘤粘蛋白抗原MUC 1和CA 125，已知这些分子的生物学特性取决于它们的O-糖基化状态。这些研究将为合理预测位点特异性O-聚糖结构提供基本工具。