Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Am Chem Soc. 2011 Nov 23;133(46):18975-91. doi: 10.1021/ja208390n. Epub 2011 Nov 1.
Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on an antibody's effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substrates, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q) were able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. Surface plasmon resonance (SPR) binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc glycoforms thus provide a useful tool for elucidating how a fine Fc N-glycan structure precisely affects the function of the Fc domain.
结构明确的 IgG-Fc 糖型是深入了解糖基化对抗体效应功能影响所急需的。本文报道了一系列均一 IgG-Fc 糖型的化学酶法合成及 Fcγ 受体结合。化学酶法包括作为供体底物的特定 N-糖基化唑啉的化学合成、在 α-甘露糖苷酶抑制剂 kifunensine 存在下在 CHO 细胞系中表达 Fc 结构域以及糖苷酶催化的合成糖唑啉对去糖基化 Fc 结构域（GlcNAc-Fc 同源二聚体）的糖基化。从节杆菌（Arthrobacter protophormiae）（Endo-A）的酶被发现对各种修饰的 N-糖基化核心唑啉非常有效，包括含有双分叉糖的衍生物，可用于 Fc 糖基化重塑，从而形成相应的均一 Fc 糖型。然而，无论是 Endo-A 还是粘帚霉内切糖苷酶突变体（EndoM-N175A 和 EndoM-N175Q）都无法将全长复合 N-糖基转移到 Fc 结构域，这表明这两种酶在 Fc 糖基化重塑方面存在局限性。通过表面等离子体共振（SPR）与合成 IgG-Fc 糖型的结合研究明确证实，双分叉 GlcNAc 部分的存在可以显著增强 Fc 与激活型 Fcγ 受体 FcγRIIIa 的结合，而与 Fc 核心岩藻糖基化无关。有趣的是，带有异常双分叉糖部分（如甘露糖或 LacNAc 部分）的 Fc 糖型也显示出与 FcγRIIIa 更强的亲和力。另一方面，双分叉 GlcNAc 或核心岩藻糖基化对 Fc 与抑制型 Fcγ 受体 FcγRIIb 的亲和力影响很小。我们的实验数据还表明，五糖 Man3GlcNAc2 核心中的α-连接甘露糖残基对于维持 Fc 与 FcγRIIIa 和 FcγRIIb 的高亲和力至关重要。因此，合成的均一 Fc 糖型为阐明精细的 Fc N-糖结构如何精确影响 Fc 结构域的功能提供了有用的工具。
