Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor.
Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor.
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DOI：
10.1021/ja208390n
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发表时间：
2011-11-23
影响因子：
15
通讯作者：
Wang LX
中科院分区：
文献类型：
--
作者：
Zou G;Ochiai H;Huang W;Yang Q;Li C;Wang LX
为了了解糖基化对抗体效应子功能的影响，非常需要结构明确的 IgG-Fc 糖型。我们在本文中报道了一系列均质 IgG-Fc 糖型的化学酶合成和 Fcγ 受体结合。化学酶法包括化学合成指定的 N-聚糖恶唑啉作为供体底物，在 α-甘露糖苷酶抑制剂 kifunensine 存在的情况下在 CHO 细胞系中表达 Fc 结构域，以及用合成聚糖恶唑啉对去糖基化的 Fc 结构域（GlcNAc-Fc 同二聚体）进行糖苷内切酶催化的糖基化。研究发现，来自原节杆菌 (Endo-A) 的酶能够非常有效地采用各种修饰的 N-聚糖核心恶唑啉（包括平分含糖衍生物）进行 Fc 糖基化重塑，从而形成相应的同质 Fc 糖型。然而，Endo-A 和冬毛霉内切糖苷酶突变体（EndoM-N175A 和 EndoM-N175Q）均无法将全长复合型 N-聚糖转移至 Fc 结构域，这表明这两种酶在 Fc 糖基化重塑中的局限性。对合成 IgG-Fc 糖型的 SPR 结合研究明确证明，平分 GlcNAc 部分的存在可以显着增强 Fc 与 FcγRIIIa（激活 Fcγ 受体）的结合，而与 Fc 核心岩藻糖基化无关。有趣的是，携带不寻常的平分糖部分（例如甘露糖或 LacNAc 部分）的 Fc 糖型也表现出与 FcγRIIIa 的亲和力增强。另一方面，平分 GlcNAc 或核心岩藻糖基化的存在对 Fc 与抑制性 Fcγ 受体 FcγRIIb 的亲和力几乎没有影响。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，合成的同质 Fc 糖型为阐明精细 Fc N-聚糖结构如何精确影响 Fc 结构域的功能提供了有用的工具。
Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on 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 substratess, 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), was able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. 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.
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