Origin of N-Glycan Site-Specific Heterogeneity

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界面， 影响细胞分化、生理和病理。关于聚糖多样性是如何 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， 在给定糖蛋白的每个位点上的聚糖结构的集合。在界定 潜在的“规则”，规定糖蛋白的选择性、位点特异性修饰，包括：1）破译 单个糖酶活性位点如何作为模板来指定区域特异性底物识别，以及2） 确定单个糖基化位点（糖位点）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多样性的途径。我们组建了一个综合研究小组 在糖酶学、重组糖蛋白表达、糖分析化学、蛋白质 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， 专业知识，以确定控制位点特异性聚糖多样性的基本特征。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 研究与供体类似物和合成聚糖受体复合的酶，并利用生物信息学 分析以产生关于糖酶底物识别进化的新假设， 的特异性这些假设将通过诱变，蛋白质重新设计和酶活性进行测试， 受体基质在目标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的糖肽检测酶促修饰的效率， 映射方法。将使用相应报告糖蛋白的结构数据进行比较 对单个糖位点进行空间限制的糖位点修饰。关于糖基化的假设 可接近性将通过诱变酶活性位点和酶活性位点上糖位点侧翼的区域来测试。 糖蛋白报告基因。在目标3中，我们将通过报告基因来检验我们关于位点特异性聚糖修饰的假设。 在培养的细胞中表达。哺乳动物中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，以确定从体外研究中确定的生物合成“规则”是否将延伸 在细胞分泌途径的更复杂环境中的聚糖修饰。拟议 研究将提供关于糖酶如何作为模板用于创建多种酶的基本知识。 聚糖结构以及其底物的空间限制如何调节这些特异性以提供 在单个聚糖位点上的可预测的聚糖多样性。

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.