Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, 467-8603, Japan.
Exploratory Research Center On Life and Living Systems (ExCELLS) and Institute for Molecular Science (IMS), National Institutes of Natural Sciences, Aichi, 444-8787, Japan.
Biomol NMR Assign. 2021 Apr;15(1):187-192. doi: 10.1007/s12104-020-10004-5. Epub 2021 Jan 10.
The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each C2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two C2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54 kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.
免疫球蛋白 G（IgG）的 Fc 部分通过与 Fcγ 受体和补体成分 C1q 相互作用，在免疫系统中促进防御效应功能。这些相互作用主要依赖于每个 C2 结构域的 Asn297 处的 N-糖基化，其中双天线复合型寡糖主要由于非还原末端半乳糖残基的存在或不存在而产生微观异质性。Fc 的晶体结构表明，一对 N-聚糖位于两个 C2 结构域之间。在这里，我们应用哺乳动物细胞代谢同位素标记技术，对 54 kDa 分子量的小鼠 IgG2b-Fc 糖型进行溶液结构表征。基于 N-聚糖以及 Fc 多肽骨架的光谱分配，我们探测了 N-聚糖修剪，特别是酶法去半乳糖基化引起的 Fc 构象变化。结果表明，去半乳糖基化通过糖蛋白相互作用的重排对 Fc 区造成结构干扰。IgG2b-Fc 糖蛋白的光谱分配将为其在溶液中与效应分子的动态构象和相互作用的 NMR 研究提供基础。
