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.

项目摘要 细胞表面和分泌的糖蛋白与细胞外环境形成复杂的接口 影响细胞分化，生理和病理学。关于聚糖多样性的知识知之甚少 受控以在不同的细胞类型或单个糖蛋白上产生不同的聚糖结构集。 聚糖结构是通过产生异质性的糖基转移酶（GTS）的作用来合成的 给定糖蛋白的每个位点上的聚糖结构的集合。挑战仍然在定义 基本的“规则”指定了糖蛋白的选择性，特定于网站的修饰，包括：1）解密 单个糖酶活性位点如何充当模板以指定型型底物识别和2） 确定单个糖基化位点（糖材）的上下文和空间约束如何限制或 限制获取产生的聚糖结构的多样性。我们组建了一个综合研究团队 具有糖酶学，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，以利用我们独特的工具集和 专业知识以确定针对特定地点聚糖多样性的基本特征。我们的目标包括（目标1） 确定甘油酶的活性位点如何为聚糖修饰提供模板。我们将追求结构 研究与供体类似物和合成聚糖受体复合物中的酶的研究并利用生物学 分析以产生有关甘油酶底物识别和 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 受体基板。在AIM 2中，我们将确定糖蛋白特异性修饰的结构基础 通过使用基于MS的糖肽来检查酶促修饰的效率，通过检查受体 映射方法。各个记者糖蛋白的结构数据将用于比较 糖材料修饰具有个体糖材料的空间约束。关于糖材的假设 可访问性将通过酶的活性位点和侧面糖材料的区域的诱变进行测试。 糖蛋白记者。在AIM 3中，我们将对记者进行特定地点的聚糖修饰测试假设 在培养细胞中的表达。在哺乳动物的糖蛋白报告中产生的位点特异性糖型 将检查分泌途径，以确定从体外研究中鉴定出的生物合成'规则是否会延长 在细胞分泌途径更复杂的环境中进行聚糖修饰。提议 研究将提供有关糖酶如何作为创建多样化的模板的基本知识 聚糖结构以及其基材的空间约束如何调整这些特异性以提供 各个聚糖站点上可预测的聚糖多样性。