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Distinct Fc? receptor N-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies.

ABSTRACT: Human immunoglobulin A (IgA) is the most prevalent antibody class at mucosal sites with an important role in mucosal defense. Little is known about the impact of N-glycan modifications of IgA1 and IgA2 on binding to the Fc? receptor (Fc?RI), which is also heavily glycosylated at its extracellular domain. Here, we transiently expressed human epidermal growth factor receptor 2 (HER2)-binding monomeric IgA1, IgA2m(1), and IgA2m(2) variants in Nicotiana benthamiana ?XT/FT plants lacking the enzymes responsible for generating nonhuman N-glycan structures. By coinfiltrating IgA with the respective glycan-modifying enzymes, we generated IgA carrying distinct homogenous N-glycans. We demonstrate that distinctly different N-glycan profiles did not influence antigen binding or the overall structure and integrity of the IgA antibodies but did affect their thermal stability. Using size-exclusion chromatography, differential scanning and isothermal titration calorimetry, surface plasmon resonance spectroscopy, and molecular modeling, we probed distinct IgA1 and IgA2 glycoforms for binding to four different Fc?RI glycoforms and investigated the thermodynamics and kinetics of complex formation. Our results suggest that different N-glycans on the receptor significantly contribute to binding affinities for its cognate ligand. We also noted that full-length IgA and Fc?RI form a mixture of 1:1 and 1:2 complexes tending toward a 1:1 stoichiometry due to different IgA tailpiece conformations that make it less likely that both binding sites are simultaneously occupied. In conclusion, N-glycans of human IgA do not affect its structure and integrity but its thermal stability, and Fc?RI N-glycans significantly modulate binding affinity to IgA.

SUBMITTER: Goritzer K

PROVIDER: S-EPMC6755811 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Distinct Fcα receptor N-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies.

Göritzer Kathrin K Turupcu Aysegül A Maresch Daniel D Novak Jan J Altmann Friedrich F Oostenbrink Chris C Obinger Christian C Strasser Richard R

The Journal of biological chemistry 20190730 38

Human immunoglobulin A (IgA) is the most prevalent antibody class at mucosal sites with an important role in mucosal defense. Little is known about the impact of N-glycan modifications of IgA1 and IgA2 on binding to the Fcα receptor (FcαRI), which is also heavily glycosylated at its extracellular domain. Here, we transiently expressed human epidermal growth factor receptor 2 (HER2)-binding monomeric IgA1, IgA2m(1), and IgA2m(2) variants in Nicotiana benthamiana ΔXT/FT plants lackin ...[more]

PMID: 31362986

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Project description: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? 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 ?-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?RIIIa, the activating Fc? 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 Fc?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? receptor, Fc?RIIb. Our experimental data also showed that the ?-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both Fc?RIIIa and Fc?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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Distinct Fc? receptor N-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies.

Publications

Distinct Fcα receptor <i>N</i>-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies.

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