A Strategy for the Synthesis of N-Linked Glycoproteins

N-连接糖蛋白的合成策略

• 批准号: 7082021
• 负责人: Clay Samuel Bennett
• 金额: $ 4.6万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082021
• 关键词: Escherichia coli; N acetylglucosamine; N acetylglucosaminidase; aminoacid tRNA ligase; asparagine; combinatorial chemistry; directed evolution; genetic library; genetic screening; glycoproteins; glycosylation; method development; mutant; oligosaccharides; peptide chemical synthesis; peptide library; postdoctoral investigator; site directed mutagenesis

DESCRIPTION (provided by applicant): Glycosylation of proteins plays a major role in a number of processes ranging from protein folding and recognition to diseases such as cancer. Despite its importance there is very little known about the effects of protein glycosylation at the molecular level. This is due to the difficulties associated with isolating homogenous glycoproteins. The following proposal will describe a new approach to the synthesis of homogenous N-linked glycoproteins. The first part of this approach involves evolving a mutant tRNA synthetase to charge a mutant tRNA with the amino acid N-acetyglucosamine-asparagine (GlcNAc-Asn) in order to incorporate it into proteins in vivo. The proposal will then describe the evolution of a glucosaminidase with enhanced transglycosylation properties, to be used to transfer complex carbohydrates to proteins containing the GlcNAc-Asn moiety. Finally, application of the automated one-pot oligosaccharide synthesis to the construction of a complex carbohydrate substrate for the mutant endoglucosaminidase will be described. If successful the combination of the methods outlined above will provide a new strategy for the efficient construction of libraries of homogenous N-linked glycoproteins.

描述（由申请人提供）：蛋白质的糖基化在从蛋白质折叠和识别到疾病（如癌症）的许多过程中起主要作用。尽管它的重要性，但在分子水平上对蛋白质糖基化的影响知之甚少。这是由于分离同质糖蛋白的困难。以下建议将描述一种合成同质N-连接糖蛋白的新方法。该方法的第一部分涉及进化突变型tRNA合成酶，以使突变型tRNA与氨基酸N-乙酰葡糖胺-天冬酰胺（GlcNAc-Asn）结合，以便将其掺入体内蛋白质中。然后，该提案将描述具有增强的转糖基化特性的氨基葡萄糖苷酶的演变，用于将复杂的碳水化合物转移到含有GlcNAc-Asn部分的蛋白质中。最后，将描述自动化一锅法寡糖合成应用于构建用于突变体内切氨基葡萄糖苷酶的复杂碳水化合物底物。如果成功，上述方法的组合将为高效构建同质N连接糖蛋白库提供一种新策略。