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 - 细胞相互作用，靶向蛋白质，调节炎症和免疫反应以及肿瘤和转移中的重要生物学作用。许多肿瘤抗原是粘蛋白样糖蛋白。 O-糖基化结构域扮演着关键的角色，取决于其扩展结构和通过一系列聚糖结构装饰的能力。 PPGALNAC转移酶家族将第一个糖A-GalNAC添加到肽核，同时通过向GalNAC添加糖来延长聚糖链。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。尽管肽序列和邻近组糖基化作用均可能很重要，但对O-糖基化调节的调节背后的机制却鲜为人知。这项工作的目的旨在详细表征PPGALNAC转移酶家族的肽底物特异性，以及添加糖（β-GAL（1-3），β-GICNAC（1-3），Alpha-Neunac（2-6））的核心1，Core 3和siAllyl转移酶。在AIM 1中，将确定一系列PPGALNAC转移酶针对几个高分子量apo-麦酸粘菌域和较小的随机肽底物的位点特异性糖基化动力学。实验性的Apo-核蛋白糖基化动力学将适合融合相邻糖基化和序列上下文效应的动力学模型。在AIM 2中，将研究在所有PPGALNAC转移酶中发现的Ricin样凝集素结构域的作用，以及小分子竞争者对PPGAINAC转移酶特定糖基化的影响。在AIM 3中，将确定并按上述确定并进行动力学建模的转移酶的位点特异性糖基化动力学。这项工作的结果是，我们将对肽序列和局部环境对O-聚糖生物合成初始步骤的作用的作用有了了解。这将使人们更好地了解生物学上重要的分子的O-糖基化，例如CD8，CD45，IGA1，PSGL-1和肿瘤粘蛋白抗原MUC1和CA125，这些分子的生物学特性依赖于其O-糖基化状态。这些研究将造成对现场特定O-Glycan结构进行合理预测的基本工具。