Origin of N-Glycan Site-Specific Heterogeneity

项目总结 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。关于多糖多样性是如何变化的，人们知之甚少 被控制以在不同的细胞类型或单个糖蛋白上产生不同的糖链结构。 糖链结构是通过糖基转移酶(Gts)的作用合成的。 给定糖蛋白每个部位上的糖链结构的集合。在定义 规定糖蛋白选择性、位点特异性修饰的潜在规则，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和2) 确定单个糖基化位点(糖位)的上下文和空间约束如何限制或 限制访问以调整产生的糖链结构的多样性。我们组建了一个综合研究小组 在糖酶、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-酶法多聚糖合成，以利用我们独特的工具箱和 识别支配特定部位的多糖多样性的基本特征的专业知识。我们的目标包括(目标1) 确定糖酶活性部位如何为糖链修饰提供模板。我们将推行结构性改革 供体类似物、合成糖链受体与生物信息学的复合酶研究 分析以产生关于糖酶底物识别和进化的新假说 专一性。这些假说将通过突变、蛋白质重新设计和酶活性来验证。 受体底物。在目标2中，我们将确定糖蛋白定点修饰的结构基础。 通过使用MS糖肽检测酶修饰受体的效率 测绘方法。将使用各自报告糖蛋白的结构数据来比较 对单个糖位进行空间约束的糖位修饰。关于糖基化的假说 可获得性将通过突变酶活性部位和位于糖基两侧的区域进行测试 糖蛋白记者。在目标3中，我们将测试我们的假设，通过记者对特定部位的多糖进行修饰 在培养的细胞中表达。哺乳动物糖蛋白报告上产生的位点特异性糖形式 将对分泌途径进行检查，以确定从体外研究中确定的生物合成规则是否会延伸 到更复杂的细胞分泌途径环境中的糖链修饰。建议数 研究将提供糖酶如何作为模板来创建多样化的基础知识 多聚糖结构及其底物的空间约束如何调节这些特性以提供 可预测的糖链在单个糖链位点上的多样性。

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.