N-蛋白糖基化是最复杂的蛋白翻译后修饰现象之一，细胞中的N-糖蛋白存在着巨大的不均一性/多样性，包括糖基化位点不均一性和N-glycan结构的不均一性。当前，尽管蛋白糖链的分析技术在已经获得了长足的进步，然而，不同结构N-glycan功能的研究却远远滞后。2015年，我们结合有机合成和酶催化发展了对结构非对称N-glycan的CSEE合成方法。于2017年利用我们课题组改造的新型N-糖基转移酶ApNGTQ469A，协同GlcN乙酰基转移酶GlmA，进一步开发了合成N-GlcNAc修饰糖肽/蛋白的方法，并合成了一系列N-GlcNAc修饰多肽。本申请中，我们将合成高丰度的100种人N-glycan物质库。并利用酶法将其连接到GlcNAc修饰的IFNβ上，系统地测试不同N-glycan结构对IFNβ活性和稳定性的影响。

N-Glycosylation is one of the most prominent post-translational protein modifications and plays a role in various cellular functions including protein folding, sorting, and stability andcell-cell interactions. Naturally occurring N-glycoproteins exhibit glycoform heterogeneity with respect to N-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). Biofunctional studies of N-glycans on proteins thus remain challenging tasks due to the complexity, diversity and low abundance of these glycans. We have developed an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse N-glycans..More recently, our group developed a novel enzymatic approach for facile production of glycopeptides carrying natural eukaryotic N-glycans. In this approach, peptides can be GlcNAcylated at one or two natural N-glycosylation sites via two-step enzymatic reactions catalyzed by an evolved N-glycosyltransferase (ApNGTQ469A) and a glucosamine N-acetyltransferase (GlmA), respectively. The resulting GlcNAc-peptides were further modified by an endo-b-N-acetylglucosaminidase M mutant (EndoMN175Q) to generate glycopeptides. In three steps of enzymatic catalysis, glycopeptides carrying complex-type N-glycans can be efficiently synthesized. In this application, we plan to synthesize 100 abundant N-glycans, which will be conjugated to Interferon β. Next, the activity and stability of the corresponding glycoprotein will be systematically investigated.