Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

项目总结/摘要 该研究的目的是开发简便的化学酶促方法来合成 具有生物医学意义的同质N-糖蛋白。功能糖组学研究中的一个主要问题 和糖蛋白治疗应用的障碍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法，利用转糖基化 一类内切糖基化酶（ENGases）的活性，其使得能够在游离聚糖和葡聚糖之间进行“天然连接”。 GlcNAc标记的蛋白质形成具有天然糖苷键的同质糖蛋白。我们已经发现 能够利用高活性聚糖的基于内切糖苷酶的新突变体糖苷酶 恶唑啉用于转糖基化，但缺乏产物水解活性。糖苷酶催化的天然 连接允许糖和蛋白质部分的独立操作， 糖蛋白组装的方法。该方法已成功地应用于合成一系列的 复合糖肽如HIV-1糖肽抗原和CD 52糖蛋白。还已经 探索重组糖蛋白的聚糖重塑，包括人促红细胞生成素（EPO）和 治疗性抗体在这项应用中，我们的目的是改进化学酶法，扩大其 范围，并加快其作为合成均质糖型的通用方法的应用， 糖蛋白为实现这些目标，将努力实现四个具体目标。前两个目标的重点是 从细菌内切糖苷酶产生新的内切糖苷酶，以及从一系列 第三个目的是开发一种具有不同的（β 1，6，β 1，3/1，4和β 1，2）-岩藻糖苷键的E.杆菌 共表达系统，以产生含有GlcNAc和Glc的蛋白质，这些蛋白质将作为 糖基化治疗性蛋白质的组合合成，其目的是提高 第四个目的是建立一种用于溶酶体糖基化重塑的方法， 酶如具有甘露糖-6-磷酸（M6 P）寡糖的重组人β-葡糖苷酶 用于在酶替代疗法中改善它们的细胞摄取。圆满完成拟议的 研究将显着扩大化学酶法的范围;提供新的使 技术的糖生物学和生物技术的社区;并加快应用的 用于改善治疗性蛋白质功效的化学酶促方法。

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.