Covergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

糖肽和糖蛋白的 Covergent 化学酶法合成

• 批准号: 9066700
• 负责人: LAI-XI WANG
• 金额: $ 29.15万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9066700
• 关键词: Acids; Address; Affect; Antibodies; Biological; Biological Process; Cell Adhesion; Chemicals; Complex; Development; Endoglycosidases; Engineering; Enzymes; Evaluation; Event; Funding; Generations; Glucose; Glycobiology; Glycogen storage disease type II; Glycopeptides; Glycoproteins; Goals; Grant; Half-Life; Health; Heterogeneity; Human; Hybrids; Hydrolysis; Immunoglobulin G; Kinetics; Knowledge; Ligation; Methods; Modeling; Molecular; Monosaccharides; Multienzyme Complexes; N acetylglucosaminidase; Oligosaccharides; Pathway interactions; Peptides; Play; Polysaccharides; Post-Translational Protein Processing; Protein Glycosylation; Proteins; Reaction; Research; Role; Serum; Site-Directed Mutagenesis; Source; Speed; Structure; Substrate Specificity; System; Testing; Therapeutic; analog; base; chemical synthesis; enzyme replacement therapy; expression cloning; gain of function; glucosidase; glycosylated IgG; glycosylation; glycosyltransferase; mannose 6 phosphate; mutant; novel; pathogen; polypeptide; programs; protein function; protein structure; receptor binding; sugar; tool; tumor progression; uptake

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop a general and highly efficient chemo enzymatic method for making diverse homogeneous glycopeptides and glycoproteins of biomedical significance. A major problem in functional glycomics studies and glycoprotein therapeutic applications is the lack of efficient methods to produce glycan-defined glycoproteins. We have recently developed a chemo enzymatic method that exploits the trans glycosylation activity of a class of endoglycosidases (ENGases) that enables the "native ligation" between free glycan and GlcNAc- tagged protein to form homogeneous glycoproteins with native glycosidic linkage. We found that synthetic glycan oxazoline and ENGase-based glycosynthase that we created is an excellent pair for an efficient transglycosylation. This method permits independent manipulations of the sugar and protein portions, providing a highly convergent and potentially general approach to glycoprotein assembly. In this renewal application, we aim at expanding the scope of the chemo enzymatic method and speeding up its application by pursuit of the following four specific aims. Aim 1 is to expand the synthetic repertoire by evaluating new enzymes, mutants, and distinct donor and acceptor substrates for glycoprotein synthesis. Aim 2 is to determine the crystal structures of EndoS and EndoF3, and their complexes with substrates/substrate analogs, which will provide a molecular level understanding of how these ENGases differentially recognize the substrates in hydrolysis and transglycosylation. Aim 3 is to explore a two-step enzymatic strategy for direct glycosylation of polypeptides and proteins, including the study of N-glycosyltransferase (NGT) for directly introducing a monosaccharide primer into polypeptide and the subsequent extension of the sugar chains via ENGase-catalyzed transglycosylation. Aim 4 is to remodel lysosomal enzymes with synthetic mannose 6-phosphate glycans aiming to enhance the therapeutic efficiency in enzyme replacement therapy (ERT). The proposed study is expected to provide important new tools in chemo enzymatic synthesis and the knowledge gained will speed up glycoprotein therapeutic applications.

描述（由申请人提供）：拟议研究的目标是开发一种通用且高效的化学酶促方法，用于制备具有生物医学意义的多种均质糖肽和糖蛋白。功能糖组学研究和糖蛋白治疗应用中的一个主要问题是缺乏生产聚糖定义的糖蛋白的有效方法。我们最近开发了一种化学酶法，该方法利用一类糖苷内切酶（ENGase）的转糖基化活性，使游离聚糖和 GlcNAc 标记蛋白之间能够“天然连接”，形成具有天然糖苷键的同质糖蛋白。我们发现，我们创建的合成聚糖恶唑啉和基于 ENGase 的糖合酶是高效转糖基化的绝佳组合。该方法允许独立操作糖和蛋白质部分，为糖蛋白组装提供高度收敛和潜在通用的方法。在本次更新申请中，我们旨在通过追求以下四个具体目标来扩大化学酶法的范围并加速其应用。目标 1 是通过评估糖蛋白合成的新酶、突变体以及不同的供体和受体底物来扩展合成库。目标 2 是确定 EndoS 和 EndoF3 的晶体结构，以及它们与底物/底物类似物的复合物，这将提供对这些 ENGase 如何在水解和转糖基化中差异识别底物的分子水平理解。目标3是探索一种用于多肽和蛋白质直接糖基化的两步酶促策略，包括研究N-糖基转移酶（NGT）将单糖引物直接引入多肽中，以及随后通过ENGase催化的转糖基化延伸糖链。目标 4 是用合成甘露糖 6-磷酸聚糖重塑溶酶体酶，旨在提高酶替代疗法 (ERT) 的治疗效率。拟议的研究预计将为化学酶合成提供重要的新工具，所获得的知识将加速糖蛋白治疗应用。