Origin of N-Glycan Site-Specific Heterogeneity

N-聚糖位点特异性异质性的起源

• 批准号: 10063537
• 负责人: Natarajan Kannan
• 金额: $ 84.55万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063537
• 关键词: 3-Dimensional; Active Sites; Address; Anabolism; Analytical Biochemistry; Analytical Chemistry; Architecture; Automobile Driving; Biochemical; Bioinformatics; Biological; Biological Process; Catalysis; Cell Adhesion; Cell Line; Cell surface; Cells; Chemicals; Complex; Cultured Cells; Data; Development; Disease; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Etiology; Evolution; Family; Gene Targeting; Genetic Diseases; Genetic Transcription; Glycopeptides; Glycoproteins; Goals; Heterogeneity; Human; Human Genetics; In Vitro; Individual; Knowledge; Mass Spectrum Analysis; Methods; Modeling; Modification; Mutagenesis; Names; Outcome; Pathology; Pathway interactions; Physiology; Polysaccharides; Production; Proteins; Recombinants; Reporter; Research; Resources; Signal Transduction; Site; Specific qualifier value; Specificity; Structure; System; Testing; To specify; Translations; Wood material; Work; analog; base; bioinformatics tool; cell type; enzyme activity; enzyme structure; extracellular; glycosylation; glycosyltransferase; improved; insight; mutant; pathogen; skills; structural biology; sugar

PROJECT SUMMARY Cell surface and secreted glycoproteins form a complex interface with the extracellular environment that influences cellular differentiation, physiology, and pathology. Very little is known about how glycan diversity is controlled to produce distinct sets of glycan structures in different cell types or on individual glycoproteins. Glycan structures are synthesized by the action of glycosyltransferases (GTs) that yield heterogeneous ensembles of glycan structures on each site of a given glycoprotein. Challenges remain in defining the underlying `rules' that specify selective, site-specific modification of glycoproteins, including: 1) deciphering how individual glycoenzyme active sites act as templates to specify regiospecific substrate recognition and 2) determining how the context and steric constraints of individual glycosylation sites (glycosites) can limit or restrict access to tune the diversity of glycan structures produced. We assembled an integrated research team with expertise in glyco-enzymology, recombinant glycoprotein expression, glyco-analytical chemistry, protein structural biology, bioinformatics, and chemo-enzymatic glycan synthesis to leverage our unique toolsets and expertise to identify the essential features that govern site-specific glycan diversity. Our aims include (Aim 1) determining how glycoenzyme active sites provide templates for glycan modification. We will pursue structural studies on enzymes in complex with donor analogs and synthetic glycan acceptors and leverage bioinformatic analyses to generate new hypotheses regarding the evolution of glycoenzyme substrate recognition and specificity. These hypotheses will be tested by mutagenesis, protein redesign, and enzyme activity toward acceptor substrates. In Aim 2 we will determine the structural basis for site-specific modification of glycoprotein acceptors by examining the efficiency of enzymatic modification through the use of MS-based glycopeptide mapping approaches. Structural data for the respective reporter glycoproteins will be used to compare glycosite modification with steric constraints for individual glycosites. Hypotheses regarding glycosite accessibility will be tested by mutagenesis of enzyme active sites and regions that flank the glycosites on the glycoprotein reporters. In Aim 3 we will test our hypotheses for site-specific glycan modifications by reporter expression in cultured cells. Site-specific glycoforms produced on the glycoprotein reporters in the mammalian secretory pathway will be examined to determine if biosynthetic `rules' identified from in vitro studies will extend to glycan modifications in the more complex environment of the cellular secretory pathway. The proposed studies will provide fundamental knowledge on how glycoenzymes act as templates for the creation of diverse glycan structures and how the steric constraints of their substrates tune those specificities to provide predictable glycan diversity on individual glycan sites.

项目概要 细胞表面和分泌的糖蛋白与细胞外环境形成复杂的界面， 影响细胞分化、生理学和病理学。人们对聚糖的多样性知之甚少 控制在不同的细胞类型或单个糖蛋白上产生不同的聚糖结构。 聚糖结构是通过糖基转移酶 (GT) 的作用合成的，可产生异质性 给定糖蛋白每个位点上的聚糖结构集合。定义挑战仍然存在 指定糖蛋白的选择性、位点特异性修饰的基本“规则”，包括：1）破译 单个糖酶活性位点如何充当模板来指定区域特异性底物识别以及 2) 确定各个糖基化位点（糖基化位点）的背景和空间限制如何限制或 限制调节所产生的聚糖结构的多样性。我们组建了一个综合研究团队 拥有糖酶学、重组糖蛋白表达、糖分析化学、蛋白质方面的专业知识 结构生物学、生物信息学和化学酶聚糖合成利用我们独特的工具集和 确定控制位点特异性聚糖多样性的基本特征的专业知识。我们的目标包括（目标 1） 确定糖酶活性位点如何为聚糖修饰提供模板。我们将追求结构性 研究酶与供体类似物和合成聚糖受体的复合物并利用生物信息学 分析产生关于糖酶底物识别和进化的新假设 特异性。这些假设将通过诱变、蛋白质重新设计和酶活性来检验 受体底物。在目标 2 中，我们将确定糖蛋白位点特异性修饰的结构基础 通过使用基于 MS 的糖肽检查酶促修饰的效率来识别受体 映射方法。各个报告糖蛋白的结构数据将用于比较 对单个糖位点进行空间限制的糖位点修饰。关于糖位点的假设 可及性将通过酶活性位点和糖位点侧翼区域的诱变来测试 糖蛋白记者。在目标 3 中，我们将测试记者对位点特异性聚糖修饰的假设 在培养细胞中表达。哺乳动物糖蛋白报告基因上产生的位点特异性糖型 将检查分泌途径，以确定体外研究确定的生物合成“规则”是否会延长 细胞分泌途径的更复杂环境中的聚糖修饰。拟议的 研究将提供关于糖酶如何作为模板来创建多种酶的基础知识 聚糖结构以及其底物的空间限制如何调整这些特异性以提供 单个聚糖位点上可预测的聚糖多样性。