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Impact of N-glycosylation on Fcγ receptor / IgG interactions: unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions
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DOI：10.1080/19420862.2019.1581017.png)
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Abstract
En 中文
The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (Fc gamma R) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the Fc gamma R capture method, as well as Fc gamma R N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged Fc gamma Rs at the biosensor surface. Coil-tagged Fc gamma R capture outperformed all other capture strategies applied to the SPR study of IgG-Fc gamma R interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged Fc gamma Rs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the Fc gamma R N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.
Keywords:
Fc gamma receptors
IgG
N-glycosylation
Surface plasmon resonance
E
K coiled-coil interactions
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