A Strategy for the Synthesis of N-Linked Glycoproteins

描述(申请人提供)：蛋白质的糖基化在从蛋白质折叠和识别到癌症等疾病的许多过程中发挥着重要作用。尽管它很重要，但人们对蛋白质糖基化在分子水平上的影响知之甚少。这是由于分离均一糖蛋白的困难所致。下面的提案将描述一种合成均一的N-连接糖蛋白的新方法。这种方法的第一部分涉及进化突变的tRNA合成酶，使突变的tRNA与氨基酸N-乙酰氨基葡萄糖-天冬酰胺(GlcNAc-ASN)充电，以便将其整合到体内的蛋白质中。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进化，该酶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，将描述一锅法低聚糖的自动化合成在构建突变的内糖氨酸酶的复合碳水化合物底物中的应用。如果成功，上述方法的结合将为有效构建均一的N-连接糖蛋白文库提供一种新的策略。

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.