Establishing the Molecular Basis of Glycoconjugate Glycosylation

P R O J E C T I 这个项目是一个项目项目的一部分，旨在为高度活跃的表达提供一个平台 将多聚糖加工酶转化为新的高特异性工具，用于合成复杂多聚糖，检测 它们在细胞环境中的糖蛋白和糖脂上的存在，以及对 在病程中的再分布。这不是一项小任务，因为据估计有7000种不同的多糖 在哺乳动物系统和~200糖基转移酶(GT)有助于它们在人类中的合成。我们 将通过建立对分子水平的理解来朝着完成这项任务迈出重要的一步 GT特异性的起源。最初的焦点是GT的一个子集，该子集在修饰糖链的末端方面很重要 糖蛋白和糖脂，唾液酸基转移酶和岩藻糖基转移酶家族的成员，以及 建立一个研究其他家庭的方案。追求三个总体目标将有助于实现这一点 理解。1)GTS的底物专一性将使用糖链阵列来鉴定糖 发生初步识别的残基和连接。将开发基于酶的新技术 以实现对已识别的糖链的灵敏检测，并将结果提供给内部和外部 合作者允许开发新的合成程序和标记策略，这将进一步 展开阵列。2)GT及其结合糖供体和受体的原子能级结构 制作。将应用X射线结晶学和核磁共振方法相结合的方法，特别是 表达平台能够产生均一糖基化的GT用于 用于核磁共振研究的结晶学和稀有同位素标记的GTS。由此产生的结构将提供 合理设计功能化受体和供体类似物以及未来GT所需的信息 抑制剂和转基因酶。3)糖基转移酶的效率和特异性 将使用糖蛋白底物在细胞环境中检查反应，以评估 蛋白质背景和细胞位置对GT特异性的影响。这些信息将在以下方面至关重要 推断基于酶的试剂在体内的作用的分子水平特异性。

P R O J E C T I This project is a part of a program project directed at turning a platform for the expression of highly active glycan processing enzymes into new high-specificity tools for the synthesis of complex glycans, the detection of their existence on glycoproteins and glycolipids in cellular environments, and the monitoring of redistribution in the course of disease. This is no small task as there are an estimated 7000 distinct glycans in mammalian systems and ~200 glycosyltransferases (GTs) contributing to their synthesis in humans. We will take an important step toward accomplishing this task by building a molecular level understanding ofthe origin of GT specificity. The initial focus is a subset of GTs important in modifying the termini ofthe glycans on glycoproteins and glycolipids, members ofthe sialyltransferase and fucosyltransferase families, and building a protocol for the study of other families. Pursuit of three general aims will contribute to this understanding. 1) Substrate specificity of GTs will be characterized using glycan arrays to identify sugar residues and linkages where primary recognition occurs. Novel enzyme based technology will be developed to allow sensitive detection of recognized glycans and the results will be fed to internal and external collaborators to allow development of new synthetic procedures and tagging strategies that will further expand arrays. 2) Atomic level structures of GTs and their bound sugar donors and acceptors will be produced. A combination of X-ray crystallography and NMR methodology will be applied, taking particular advantage ofthe expression platform's ability to produce homogeneously glycosylated GTs for crystallography and sparse isotope labeled GTs for NMR investigation. The resulting structures will provide information needed forthe rational design of functionalized acceptor and donor analogs, as well as future GT inhibitors and genetically modified enzymes. 3) The efficiency and specificity of glycosyltransferase reactions will be examined in cellular environments using a glycoprotein substrate in order to assess the influence of a protein context and cellular location on GT specificity. The information will be essential in extrapolating molecular level specificities to action of enzyme-based reagents in vivo.