期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 133, 期 46, 页码 18975-18991出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja208390n
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资金
- National Institutes of Health (NIH) [R01 GM096973, R01 GM080374]
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 Fc gamma 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 alpha-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 Fc gamma RIIIa, the activating Fc gamma receptor, independent of Fc core-fucosylation. Interestingly, the Pc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to Fc gamma RIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fc gamma receptor, Fc gamma RIIb. Our experimental data also showed that the alpha-linked mannose residues in the pentasaccharide Man3G1cNAc2 core was essential to maintain a high affinity of Fc to both Fc gamma RIIIa and Fc gamma RIIb. 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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