固相酶催化法组装人源化N-寡糖的研究

As one of the important biological molecule, sugar (carbohydrate) widely exists in living organisms. Carbohydrates can covalently link with other substrates such as peptides and lipids to generate the glycoconjugates, which are involved in almost every biological pathway. Glycoprotein is the most abundant glycoconjugate, and most of the secretory or membrane proteins in eukaryotic cells are glycosylated. In human bodies, glycans which modify proteins regulate their physiological properties and are closely related with the cellular differentiation, apoptosis, carcinogenesis, autoimmunization and signal transduction. In order to investigate the biological function of glycan and its effects on glycoproteins, a useful oligosaccharide library with various human-type glycan structures is absolutely desired. In this study, we propose a novel solid-phase enzymatic method for synthesizing various human N-glycans. In our strategy, for mimicking the dolichol-linked oligosaccharides (DLO) synthetic pathway in the endoplasmic reticulum (ER), a lipid-linked chitobiose is first immobilized onto a solid-phase carrier, and the glycan chain would be elongated by a series of recombinant glycosyltransferases (GTases) selected from the eukaryotic N-glycosylation pathway. Our method will produce various human N-glycans with significant amount by simply shifting the GTases, providing useful lipid-linked N-glycans for scientific studies of glycoproteins and pharmaceutical applications.

糖广泛地存在于生物体内，是维持生命活动的重要物质之一。它单独，或与其他生命物质形成糖聚合物参与生命体中的几乎每一个生理活动。糖蛋白是最为普遍的一类糖聚合物，其中与多肽共价结合的糖链能有效地修饰蛋白质结构、调控其生理活性，并参与细胞分化和凋亡、癌变、自身免疫等重要生理过程。为研究糖链的生物活性及其对蛋白质的修饰调控机制，开发一种稳定地获得特定糖链的方法具有重要的意义。本课题拟简化真核细胞多萜醇寡糖（DLO）前体的生物合成途径，通过体外大量表达合成人源糖链所需的糖基转移酶，结合固相合成的方法，制定一种新的固相酶促寡糖合成的策略，旨在实现人源脂肪醇寡糖（N-糖链）的大量且结构可控性生产。本研究将应用于糖蛋白生理活性以及调控机制等的研究，并为生产人源化糖蛋白药物和制备寡糖抗原等提供新的策略。

糖广泛地存在于生物体内。它单独，或与其他生命物质形成糖聚合物参与生命体中的几乎每一个生理活动。为研究糖链的生物活性及其对蛋白质的修饰调控机制，开发一种稳定地获得特定糖链的方法具有重要的意义。本课题以真核细胞多萜醇寡糖（DLO）前体的生物合成途径为模板，通过体外大量表达合成人源糖链所需的糖基转移酶，结合固相合成的方法，制定一种新的固相酶促寡糖合成的策略，实现了人源复合型糖链 Sial2Gal2GlcNAc2Man3GlcNAc2以及人源生物标志物人源生物标记物 SialGalGlcNAc2的大量且结构可控性生产。本研究将应用于糖蛋白生理活性以及调控机制等的研究，并为生产人源化糖蛋白药物和制备寡糖抗原等提供新的策略。