Development of streamlined chemoenzymatic glycan remodeling systems for antibodies and other important glycoproteins

项目摘要/摘要 这项提案的目标是开发高效和简化的葡聚糖低酶系统。 抗体和其他重要糖蛋白的重塑，通过酶固定化，可伸缩的多糖 生产，以及试剂盒的开发。这样的固定化酶，当与激活的糖链文库结合时， 试剂盒，可以帮助一般的学术和工业用户，特别是非专业用户，准备由多糖定义的 用于结构和功能研究的糖蛋白。简化的方法也可以应用于扩大规模 制备具有治疗潜力的均一糖蛋白。蛋白质糖基化是最重要的 无处不在的翻译后修饰。它深刻地影响蛋白质的性质，如在体内的折叠 稳定性、免疫原性和药代动力学，也直接参与许多重要的生物 过程，包括细胞黏附、癌症进展、宿主-病原体相互作用和免疫反应。 糖蛋白研究中的一个主要问题是结构的异质性和分离的困难。 均一糖形式，用于详细的结构和功能研究。尽管取得了重大进展， 用于生产糖蛋白，如全化学合成和生物合成途径 在不同的宿主表达系统中进行工程，所能获得的纯糖形式仍然相当有限。 为了解决这一挑战，一种新的化学酶方法生产均一的糖蛋白和 糖肽是近几年发展起来的一种生物制剂。这种收敛的方法由两个关键步骤组成：去糖基化 具有内切糖苷酶的糖蛋白，然后将所需的激活的N-糖链附着到 一种新的糖合酶的蛋白质-GlcNAc受体。此SBIR II应用程序建立在阶段成功的基础上 我研究了(1R43GM134816-01)，在那里我们成功地完成了内切糖苷酶S2的固定化 (EndoS2)及其相应的糖合成酶突变体EndoS2-D184M。EndoS2-D184M 糖合酶的固定化是一种面向位点的方法，几乎完全保持了酶的活性。我们 已证明固定化酶对治疗性糖链重塑是高效的。 抗体。在这项第二阶段的研究中，我们建议追求以下三个具体目标。目标1是扩张 这一固定化策略与其他糖苷酶/糖合成酶相结合，建立了一个糖苷酶工具箱。 改建。目标2是优化和扩大我们针对不同恶唑类药物的生产方案，并显著 降低他们的生产成本。目标3是开发糖链重塑试剂盒并建立可扩展的糖链重塑 协议。

Project abstract/summary The objectives of this proposal are to develop efficient and streamlined cheomoenzymatic systems for glycan remodeling of antibodies and other important glycoproteins, through enzyme immobilization, scalable glycan production, and kits development. Such immobilized enzymes, when combined with activated glycan library as kits, can help general academic and industrial users, particularly non-specialists, to prepare glycan-defined glycoproteins for structural and functional studies. The streamlined approach can also be applied in scale-up preparation of homogenous glycoproteins with therapeutic potential. Protein glycosylation is one of the most ubiquitous posttranslational modifications. It profoundly affects a protein’s properties such as folding, in vivo stability, immunogenicity, and pharmacokinetics, and also directly participate in many important biological processes, including cell adhesion, cancer progression, host-pathogen interactions, and immune responses. A major issue in glycoprotein studies is the structural heterogeneity and the difficulty in isolating homogeneous glycoforms for detailed structural and functional studies. Although significant progresses have been made for producing glycan-defined glycoprotein such as total chemical synthesis and biosynthetic pathway engineering in different host expression system, the pure glycoforms that can be achieved are still quite limited. To address this challenge, a novel chemoenzymatic method to produce homogeneous glycoprotein and glycopeptides has been developed recently. This convergent approach consists of two key steps: deglcosylation of glycoproteins with an endoglycosidase and subsequent attachment of a desired activated N-glycan to the protein-GlcNAc acceptor by a novel glycosynthase. This SBIR II application is built on the success of the Phase I study (1R43GM134816-01), where we have successfully finished the immobilization of endoglycosidase S2 (EndoS2) from S. pyogenes and its corresponding glycosynthase mutant EndoS2-D184M. The EndoS2-D184M glycosynthase was immobilized by a site-oriented approach which almost fully retains the activity of enzyme. We have demonstrated that the immobilized enzymes are highly efficient for glycan remodeling of therapeutic antibodies. In this Phase II research, we propose to pursue the following three specific aims. Aim 1 is to expand this immobilization strategy to other glycosidases/glycosynthases to establish a tool box of enzymes for glycan- remodeling. Aim 2 is optimize and scale up our production protocol for different oxazolines and significantly lower their production cost. Aim 3 is to develop glycan remodeling kits and establish scalable glycan remodeling protocols.