Convergent Chemoenzymatic Synthesis of Glycopeptides and Glycoproteins

糖肽和糖蛋白的聚合化学酶合成

• 批准号: 7845309
• 负责人: LAI-XI WANG
• 金额: $ 52.5万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7845309
• 关键词: Adhesions; Affect; Area; Biological; Bos taurus structural-GP protein; Cell Adhesion; Development; Endoglycosidases; Engineering; Enzymes; Eukaryota; Event; Extracellular Domain; Funding; Glycobiology; Glycopeptides; Glycoproteins; Half-Life; Heterogeneity; Human; IgG1; Immune response; Infection; Link; Mannose; Methods; N acetylglucosaminidase; Neoplasm Metastasis; Oligosaccharides; Parents; Play; Post-Translational Protein Processing; Proteins; Recombinants; Recovery; Research; Research Project Grants; Role; Serum; Site-Directed Mutagenesis; Source; Speed; Structure; Study models; Substrate Specificity; Therapeutic; United States National Institutes of Health; base; glycosylation; mutant; novel; parent grant; pathogen; programs; protein degradation; protein structure function; public health relevance; ribonuclease B; tumor

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.

描述（由申请人提供）：糖肽和糖蛋白的会聚化学酶促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宣布恢复法案资金可用于竞争性修订申请。该研究项目的重点是开发有效的化学酶法合成具有生物医学意义的N-糖肽和N-糖蛋白。N-糖基化是真核生物中蛋白质翻译后的一种主要修饰方式。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起重要作用。共价连接的寡糖可以深刻地影响蛋白质的结构、功能及其血清半衰期。然而，糖生物学研究中的一个主要挑战是处理糖蛋白的结构异质性，糖蛋白通常以各种糖型的混合物形式产生。由于纯糖型极难从天然和重组来源中分离，因此合成已成为获得均质材料的重要来源。该基金旨在探索内切-2-N-乙酰氨基葡萄糖苷酶（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。重点介绍了底物特异性的研究、核糖核酸酶B的模型研究以及在人IgG 1-Fc糖基化工程中的初步应用。我们最近发现了用于构建同质天然N-糖蛋白的新型ENGase基糖苷酶，这是该领域的一个重大进展，在母提案中尚未提出。此外，最近解决的Endo-A的晶体结构提供了令人兴奋的新的机会，工程酶，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法的范围，并加快其在构建用于结构和功能研究的生物学重要糖蛋白方面的应用。它有两个具体目标：1）ENGases的定点诱变以提高效率并扩大化学酶法的范围; 2）化学酶法合成用于结构和功能研究的所选糖蛋白的纯糖型。这些将包括用于ER相关降解（ERAD）研究的人CD 2胞外结构域的特定糖型，以及用于NMR结构研究的核糖核酸酶B和CD 2的纯糖型。这一修订申请大大扩大了父母补助金的范围和方向。 公共卫生相关性：该竞争性修订申请旨在开发用于结构和功能研究的糖蛋白高效合成的新方法。所获得的信息对于基于糖蛋白的治疗方法的开发将是有价值的。