State Key Laboratory of Natural and Biomimetic Drugs, and Department of Chemical Biology at School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
J Am Chem Soc. 2021 Feb 24;143(7):2846-2856. doi: 10.1021/jacs.0c12448. Epub 2021 Feb 12.
-Glycosylation represents an essential type of posttranslational modification for proteins. However, deciphering the functions of -glycosylation remains a challenge due to the lack of analytical and biochemical methods to accurately differentiate the protein glycoforms with various intact glycans. Here we report our synthesis and evaluation of homogeneously glycosylated interleukin-17A (IL-17A), based on a synthetic approach combining solid-phase synthesis of (glyco)peptides, chemoenzymatic glycan modification on segments, and chemical ligations. The obtained homogeneous glycoproteins allow for the demonstration of the stabilizing role of -glycans during the folding step. A comparison of three IL-17A glycoforms in a normal human dermal fibroblast (NHDF) assay reveals dose-dependent interleukin-6-inducing activities in all cases, wherein the glycoform with sialyl undecasaccharides displays much weaker stimulatory effect than that of the GlcNAc- or GlcNAc(β)GlcNAc-modified proteins. Further surface plasmon resonance (SPR) and hydrogen/deuterium exchange mass spectroscopic experiments confirm that the evaluated complex type -glycan impedes the binding between IL-17A and its receptor IL-17RA. This structure-activity relationship study on glycoproteins highlights the viability of applying the de novo approach to probe the roles of -glycans.
糖基化是蛋白质翻译后修饰的一种重要类型。然而，由于缺乏分析和生化方法来准确区分具有各种完整聚糖的蛋白质糖型，因此破译糖基化的功能仍然是一个挑战。在这里，我们报告了基于固相合成（糖）肽、化学酶法糖基化修饰片段和化学连接的综合方法，合成和评估了均匀糖基化的白细胞介素-17A（IL-17A）。所获得的均一糖蛋白允许证明β-聚糖在折叠步骤中的稳定作用。在正常人真皮成纤维细胞（NHDF）测定中比较三种 IL-17A 糖型，在所有情况下均显示出剂量依赖性的白细胞介素-6 诱导活性，其中带有唾液酸十一聚糖的糖型的刺激作用比 GlcNAc-或 GlcNAc（β）GlcNAc 修饰的蛋白质弱得多。进一步的表面等离子体共振（SPR）和氢/氘交换质谱实验证实，所评估的复合型β-聚糖阻碍了 IL-17A 与其受体 IL-17RA 之间的结合。对糖蛋白的这种结构-活性关系研究强调了应用从头方法来探究β-聚糖作用的可行性。
