Establishing the Molecular Basis of Glycoconjugate Glycosylation

建立糖复合物糖基化的分子基础

基本信息

批准号：
9313292
负责人：
JAMES H. PRESTEGARD
金额：
$ 39.55万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9313292
关键词：
Anabolism Animals Anisotropy Bacteria Binding Biochemical Biological Biological Assay Biology Carbohydrates Catalytic Domain Cell Communication Cell physiology Cells Chemicals Chemistry Complex Coupling Crystallography Cultured Cells Data Detection Development Diagnosis Disease Environment Enzymes Family Family member Fucosyltransferase Future Geometry Glycobiology Glycoconjugates Glycolipids Glycoproteins Glycoside Hydrolases Human In Vitro Instruction Investigation Isotope Labeling Kinetics Knowledge Lanthanoid Series Elements Ligands Lipids Location Mammalian Cell Methodology Modification Molecular Molecular Chaperones Molecular Probes Monitor Nature Pathology Pathway interactions Phase Physiological Play Polysaccharides Positioning Attribute Post-Translational Protein Processing Procedures Process Production Proteins Protocols documentation Reaction Reagent Recombinants Relaxation Reporter Residual state Role Serum Sialyltransferases Signaling Molecule Specificity Spin Labels Structural Models Structure Substrate Interaction Substrate Specificity System Technology Testing Therapeutic Transfection X ray diffraction analysis X-Ray Crystallography X-Ray Diffraction analog base carbohydrate structure catalyst design enzyme model experimental study glycoprotein structure glycosylation glycosyltransferase in vivo inhibitor/antagonist member milligram novel programs sugar tool

项目摘要

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.

p r o j e c t i 该项目是一个计划项目的一部分，该项目旨在转动一个表达高度活跃的平台聚糖加工酶为合成复杂聚糖的新高特异性工具，检测它们在细胞环境中的糖蛋白和糖脂上存在，并监测在疾病过程中的重新分布。这不是很小的任务，因为估计有7000种不同的聚糖在哺乳动物系统和约200个糖基转移酶（GTS）中，有助于其在人类中的合成。我们将通过建立对分子水平的理解来迈出重要的一步 GT特异性的起源。最初的焦点是GT的子集，对于修改聚糖的末端很重要在糖蛋白和糖脂上，乙烯基转移酶和岩藻糖基转移酶家族的成员以及建立研究其他家庭的协议。追求三个一般目标将为这一点做出贡献理解。 1）GTS的底物特异性将使用聚糖阵列来表征以识别糖发生主要识别的残留和联系。将开发基于酶的新型技术允许对公认的聚糖进行敏感检测，结果将被馈送到内部和外部合作者允许制定新的合成程序和标记策略，以进一步展开数组。 2）GTS的原子级结构及其约束的供体供体和受体将是生产。将使用特定的X射线晶体学和NMR方法的组合表达平台产生同质糖基化GT的能力的优势晶体学和稀疏同位素标记为NMR研究的GTS。最终的结构将提供功能化受体和捐助者类似物的合理设计所需的信息以及未来的GT 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，以评估蛋白质环境和细胞位置对GT特异性的影响。该信息至关重要将分子水平的特异性推断到基于酶的试剂在体内的作用。