Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

描述（由申请人提供）：糖肽和糖蛋白的聚合化学酶合成此竞争性修订响应以下NIH计划：通知编号（NOT- OD-09-058）和通知标题：NIH宣布可获得竞争性修订申请的恢复法案资金。该研究项目的重点是开发有效的化学酶方法来合成具有生物医学意义的n -糖肽和n -糖蛋白。n -链糖基化是真核生物中主要的蛋白质翻译后修饰。糖蛋白在细胞粘附、肿瘤转移、病原体感染和免疫应答等许多生物学事件中发挥重要作用。共价寡糖可以深刻地影响蛋白质的结构、功能和血清半衰期。然而，糖生物学研究的一个主要挑战是处理糖蛋白的结构异质性，糖蛋白通常是由各种糖型的混合物产生的。由于纯糖型极难从天然和重组来源分离，合成已成为获得均质材料的重要来源。本基金旨在探索内切-2- n -乙酰氨基葡萄糖酶（ENGases）的转糖基化潜力，ENGases是一类特殊的内切糖苷酶，用于构建n -糖肽和n -糖蛋白。重点关注底物特异性研究、核糖核酸酶B模型研究以及在人IgG1-Fc糖基化工程中的初步应用。我们最近发现了新的基于engase的糖合酶，用于构建均匀的天然n -糖蛋白，这是在母体提案中未提出的领域取得的重大进展。此外，最近发现的endoa的晶体结构为设计酶提供了令人兴奋的新机会，以提高转糖基化效率和扩大其底物特异性。这一竞争性修订申请旨在扩大化学酶方法的范围，并加快其在构建生物学上重要的糖蛋白的结构和功能研究中的应用。它有两个具体的目的：1)酶的定点诱变，以提高效率和扩大化学酶方法的范围；2)化学酶法合成选定糖蛋白的纯糖型，用于结构和功能研究。这些将包括用于er相关降解（ERAD）研究的人类CD2细胞外结构域的特定糖型，以及用于核磁共振结构研究的核糖核酸酶B和CD2的纯糖型。这一修订申请显著扩大了家长奖助金的范围和方向。

DESCRIPTION (provided by applicant): Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins This competitive revision responds to the following NIH program: the Notice Number (NOT- OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The research project focuses on the development of efficient chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is a major posttranslational modification of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. The covalently linked oligosaccharides can profoundly affect proteins' structure, function, and their serum half-life. However, a major challenge in glycobiology research is to deal with the structural heterogeneity of glycoproteins, which are typically produced as mixtures of various glycoforms. Since pure glycoforms are extremely difficult to isolate from natural and recombinant sources, synthesis has become an essential source for obtaining homogeneous materials. The parent grant aims to explore the transglycosylation potential of endo-2-N-acetylglucosaminidases (ENGases), a special class of endoglycosidases, for constructing N-glycopeptides and N-glycoproteins. It focuses on studies of substrate specificity, model studies with ribonuclease B, and preliminary application for human IgG1-Fc glycosylation engineering. Our recent discovery of novel ENGase-based glycosynthases for the construction of homogeneous natural N-glycoproteins represents a major advance in the area that has not been proposed in the parent proposal. In addition, the recently solved crystal structure of Endo-A has provided exciting new opportunity to engineer the enzyme for enhancing the transglycosylation efficiency and for broadening its substrate specificity. This competitive revision application intends to expand the scope of the chemoenzymatic method and to speed up its application for constructing biologically important glycoproteins for structural and functional studies. It has two specific aims: 1) Site-directed mutagenesis of ENGases to enhance the efficiency and to expand the scope of chemoenzymatic method; 2) chemoenzymatic synthesis of pure glycoforms of selected glycoproteins for structural and functional studies. These will include specific glycoforms of human CD2 extracellular domain for ER-associated degradation (ERAD) studies, and pure glycoforms of ribonuclease B and CD2 for NMR structural studies. This revision application significantly expands the scope and direction of the parent grant. PUBLIC HEALTH RELEVANCE: This competitive revision application aims to develop new methods for the efficient synthesis of glycoproteins for structural and functional studies. The information gained will be valuable for development of glycoprotein-based therapeutics.