Abstract
The glycoengineering approach is used to improve biophysical properties of protein-based drugs, but its direct impact on binding affinity and kinetic properties for the glycoengineered protein and its binding partner interaction is unclear. Type I interferon (IFN) receptors, composed of IFNAR1 and IFNAR2, have different binding strengths, and sequentially bind to IFN in the dominant direction, leading to activation of signals and induces a variety of biological effects. Here, we evaluated receptor-binding kinetics for each state of binary and ternary complex formation between recombinant human IFN-β-1a and the glycoengineered IFN-β mutein (R27T) using the heterodimeric Fc-fusion technology, and compared biological responses between them. Our results have provided evidence that the additional glycan of R27T, located at the binding interface of IFNAR2, destabilizes the interaction with IFNAR2 via steric hindrance, and simultaneously enhances the interaction with IFNAR1 by restricting the conformational freedom of R27T. Consequentially, altered receptor-binding kinetics of R27T in the ternary complex formation led to a substantial increase in strength and duration of biological responses such as prolonged signal activation and gene expression, contributing to enhanced anti-proliferative activity. In conclusion, our findings reveal N-glycan at residue 25 of R27T is a crucial regulator of receptor-binding kinetics that changes biological activities such as long-lasting activation. Thus, we believe that R27T may be clinically beneficial for patients with multiple sclerosis.
Highlights
Glycosylation is the most common post-translational process in which carbohydrate moieties are attached enzymatically to protein residues to provide the stability of structure and dynamics (Mitra et al, 2006; Hanson et al, 2009; Aebi et al, 2010; Lee et al, 2015)
We performed in silico docking to elucidate the ternary complexes of R27T and IFN-β-1a with their cognate receptors, in which each receptor binds to the opposite side of the ligand
This glycan is on the proposed IFNAR2binding site, according to alanine scanning mutagenesis (Runkel et al, 2000), and this residue may be able to interact with sub-domain 1 of IFNAR2 according to the two-dimensional interaction map of the IFN-α2-IFNAR2
Summary
Glycosylation is the most common post-translational process in which carbohydrate moieties are attached enzymatically to protein residues to provide the stability of structure and dynamics (Mitra et al, 2006; Hanson et al, 2009; Aebi et al, 2010; Lee et al, 2015). The introduction of carbohydrate moieties helps impact the modification of Receptor-Binding Kinetics of R27T biophysical and pharmacological properties and overcome the inherent limitations of protein-based drugs. The N-linked glycan at residue 196 of the interleukin 5 receptor alpha subunit contributes to the ability of ligand binding and alters its biological activity (Ishino et al, 2011). Since glycosylation is highly heterogenic, owing to the factors such as attachment site and glycoforms, the understanding of how a particular site of glycosylation affects biological activity is not clear (Liu, 1992; Davis, 2002)
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