Establishing the Molecular Basis of Glycoconjugate Glycosylation

P R O J E C T I 这个项目是一个程序项目的一部分，目的是把一个高度活跃的表达平台 聚糖加工酶成为新的高特异性工具，用于合成复杂的聚糖，检测 它们存在于细胞环境中的糖蛋白和糖脂上， 疾病过程中的再分配。这不是一项小任务，因为估计有7000种不同的聚糖 在哺乳动物系统中，约有200种糖基转移酶（GT）参与其在人体中的合成。我们 将通过建立分子水平的理解，朝着完成这项任务迈出重要的一步。 GT特异性的起源。最初的焦点是在修饰聚糖末端中重要的GT的子集 糖蛋白和糖脂，唾液酸转移酶和岩藻糖基转移酶家族的成员，和 为其他家族的研究建立一个协议。追求三个总目标将有助于实现这一目标 认识1)将使用聚糖阵列表征GT的底物特异性，以鉴定糖 发生初级识别的残基和连接。将开发基于酶的新技术 允许识别聚糖的灵敏检测，结果将被馈送到内部和外部 合作者允许开发新的合成程序和标记策略， 展开数组。2)GT及其结合糖供体和受体的原子水平结构将被 制作。结合X射线晶体学和核磁共振方法将被应用，特别是 表达平台产生均匀糖基化GT的能力的优点 晶体学和稀疏同位素标记的GT用于NMR研究。由此产生的结构将提供 功能化受体和供体类似物的合理设计以及未来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， 蛋白质环境和细胞位置对GT特异性影响。这些信息将是必不可少的， 将分子水平特异性外推到基于酶的试剂在体内的作用。

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.