Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

糖肽和糖蛋白的聚合化学酶合成

• 批准号: 8627610
• 负责人: LAI-XI WANG
• 金额: $ 29.93万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8627610
• 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; 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; public health relevance; 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主要问题是缺乏有效的方法来产生聚糖定义的糖蛋白。我们最近开发了一种化学酶促方法，其利用一类内切糖苷酶（ENGase）的转糖基化活性，其使得游离聚糖和GlcNAc标记的蛋白质之间能够“天然连接”以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的糖合酶是有效转糖基化的极好配对。这种方法允许糖和蛋白质部分的独立操作，提供了一种高度收敛和潜在的通用方法糖蛋白组装。在本更新申请中，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。目的1是通过评估新的酶，突变体和不同的供体和受体底物糖蛋白合成，扩大合成库。目的2是确定EndoS和EndoF 3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ENGases如何在水解和转糖基化中差异识别底物的分子水平理解。目的3：探索多肽和蛋白质直接糖基化的两步酶促反应策略，包括利用N-糖基转移酶（NGT）直接将单糖引物引入多肽，然后通过ENGase催化的转糖基化反应延长糖链。目的4是用合成的甘露糖6-磷酸聚糖改造溶酶体酶，旨在提高酶替代疗法（ERT）的治疗效率。这项研究有望为化学酶促合成提供重要的新工具，所获得的知识将加快糖蛋白治疗应用。