Shivatare Sachin S, Shivatare Vidya S, Wu Chung-Yi, Wong Chi-Huey
Genomics Research Center, Academia Sinica.
Genomics Research Center, Academia Sinica;
J Vis Exp. 2018 Feb 5(132):55855. doi: 10.3791/55855.
We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise α-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial β-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.
我们展示了一种高效的方法，可快速制备多种常见于人类糖蛋白上的N-连接寡糖（估计超过20,000种结构）。为了实现所需的结构多样性，该策略首先通过化学酶法合成三种寡糖基氟化物模块，然后在具有关键β-甘露糖苷键的共同核心三糖的甘露糖残基的3-O和6-O位置进行逐步α-选择性糖基化。我们进一步将N-聚糖连接到氧化铝涂层玻璃（ACG）载玻片表面，以创建一个共价混合阵列，用于分析与HIV抗体的异源配体相互作用。特别是，新分离的HIV-1广谱中和抗体（bNAb）PG9与ACG阵列上紧密间隔的Man5GlcNAc2（Man5）和2,6-二唾液酸化双天线复合型N-聚糖（SCT）混合物的结合行为，为指导HIV疫苗开发的有效免疫原设计开辟了一条新途径。此外，我们的ACG阵列是研究其他HIV抗体异源配体结合行为的有力工具。
