Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

糖肽和糖蛋白的聚合化学酶合成

• 批准号: 9921425
• 负责人: LAI-XI WANG
• 金额: $ 30.8万
• 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 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9921425
• 关键词: Acids; Antibodies; Antigens; Bifidobacterium; Biological; Biotechnology; CDW52 gene; Cell Adhesion; Communities; Complex; Coupled; Development; Endoglycosidases; Enzymes; Erythropoietin; Escherichia coli; Event; Fluorides; Fucose; Fucosidase; Glycobiology; Glycogen storage disease type II; Glycopeptides; Glycoproteins; Glycosides; Goals; HIV-1; Half-Life; Heterogeneity; Human; Hydrolysis; IGF Type 2 Receptor; Immobilization; Immune response; Interferon-alpha; Ligation; Link; Methods; Modeling; Mutation; Oligosaccharides; Play; Point Mutation; Polysaccharides; Post-Translational Protein Processing; Proteins; Recombinants; Replacement Therapy; Research; Role; Series; Serum; Site; Source; Speed; Structure; System; Technology; Testing; Therapeutic; Therapeutic antibodies; Treatment Efficacy; analog; base; enzyme replacement therapy; glucosidase; glycosylation; glycosyltransferase; improved; mannose 6 phosphate; mutant; novel; pathogen; receptor binding; sugar; targeted treatment; therapeutic protein; tumor progression; uptake

Project Summary/Abstract The objectives of the proposed research are to develop facile chemoenzymatic methods for synthesizing homogeneous N-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 chemoenzymatic method that exploits the transglycosylation 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 have discovered novel endoglycosidase-based mutants, the glycosynthases, that are capable of using highly active glycan oxazolines for transglycosylation but lack the product hydrolysis activity. The glycosynthase-catalyzed native ligation permits independent manipulations of the sugar and protein portions and provides a highly convergent approach to glycoprotein assembly. This method has been successfully applied for the synthesis of a series of complex glycopeptides such as the HIV-1 glycopeptide antigens and CD52 glycoproteins. It has also been explored for glycan remodeling of recombinant glycoproteins including human erythropoietin (EPO) and therapeutic antibodies. In this application, we aim at improving the chemoenzymatic method, expanding its scope, and speeding up its application as a general method for the synthesis of homogeneous glycoforms of glycoproteins. Four specific aims will be pursued to achieve the goals. The first two aims are focused on generating new endoglycosynthases from bacterial endoglycosidases and novel -fucoligases from an array of -fucosidases with distinct (1,6, 1,3/1,4, and 1,2)-fucosidic linkages; the third aim is to develop an E. coli co-expression system to produce GlcNAc- and Glc-containing proteins that will serve as the key precursor for a combined synthesis of glycosylated therapeutic proteins with a goal of enhancing the serum half-life of therapeutic proteins; and the fouth aim is to establish a method for glycosylation remodeling of lysosomal enzymes such as the recombinant human -glucosidase with mannose-6-phosphate (M6P) oligosaccharides for improving their cellular uptake in enzymatic replacement therapy. A successful completion of the proposed research will significantly expand the scope of the chemoenzymatic method; provide new enabling technologies to the community of glycobiology and biotechnology; and speed up the applications of the chemoenzymatic method for improving the efficacy of therapeutic proteins.

项目概要/摘要 拟议研究的目标是开发简便的化学酶法来合成 具有生物医学意义的均质N-糖蛋白。功能糖组学研究的一个主要问题 和糖蛋白治疗应用的问题是缺乏有效的方法来生产聚糖定义的 糖蛋白。我们最近开发了一种利用转糖基化的化学酶方法 一类糖苷内切酶 (ENGases) 的活性，可实现游离聚糖和聚糖之间的“天然连接” GlcNAc 标记的蛋白质形成具有天然糖苷键的均质糖蛋白。我们发现 新型基于糖苷内切酶的突变体，即糖合酶，能够使用高活性的聚糖 恶唑啉可进行糖基转移，但缺乏产物水解活性。糖合酶催化的天然 连接允许独立操作糖和蛋白质部分，并提供高度会聚的 糖蛋白组装方法。该方法已成功应用于一系列的合成 复杂的糖肽，例如 HIV-1 糖肽抗原和 CD52 糖蛋白。也曾被 探索重组糖蛋白的聚糖重塑，包括人促红细胞生成素 (EPO) 和 治疗性抗体。在此应用中，我们的目标是改进化学酶法，扩展其 范围，并加速其作为合成均质糖型的通用方法的应用 糖蛋白。为了实现这些目标，将追求四个具体目标。前两个目标集中于 从细菌内切糖苷酶生成新的内切糖合酶，并从一系列的新型α-岩藻糖连接酶 具有独特的（α1,6、α1,3/1,4 和 α1,2）-岩藻糖苷键的α-岩藻糖苷酶；第三个目标是开发大肠杆菌 共表达系统产生含有 GlcNAc 和 Glc 的蛋白质，这些蛋白质将作为 联合合成糖基化治疗蛋白，目的是延长其血清半衰期 治疗性蛋白质；第四个目标是建立溶酶体糖基化重塑的方法 酶，例如带有 6-磷酸甘露糖 (M6P) 寡糖的重组人 α-葡萄糖苷酶 提高细胞对酶替代疗法的吸收。顺利完成拟议任务 研究将显着扩大化学酶法的范围；提供新的赋能 糖生物学和生物技术界的技术；并加快应用 用于提高治疗性蛋白质功效的化学酶法。