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Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding.

ABSTRACT: Immunoglobulin G1 (IgG1)-based therapies are widespread, and many function through interactions with low-affinity Fc γ receptors (FcγR). N-glycosylation of the IgG1 Fc domain is required for FcγR binding, though it is unclear why. Structures of the FcγR:Fc complex fail to explain this because the FcγR polypeptide does not bind the N-glycan. Here we identify a link between motion of the N-glycan and Fc:FcγRIIIa affinity that explains the N-glycan requirement. Fc F241 and F243 mutations decreased the N-glycan/polypeptide interaction and increased N-glycan mobility. The affinity of the Fc mutants for FcγRIIIa was directly proportional to the degree of glycan restriction (R(2) = 0.82). The IgG1 Fc K246F mutation stabilized the N-glycan and enhanced affinity for FcγRIIIa. Allosteric modulation of a protein/protein interaction represents a previously undescribed role for N-glycans in biology. Conserved features suggesting a similar N-glycan/aromatic interaction were also found in IgD, IgE, and IgM, but not IgA.

SUBMITTER: Subedi GP

PROVIDER: S-EPMC4192013 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding.

Subedi Ganesh P GP Hanson Quinlin M QM Barb Adam W AW

Structure (London, England : 1993) 20140904 10

Immunoglobulin G1 (IgG1)-based therapies are widespread, and many function through interactions with low-affinity Fc γ receptors (FcγR). N-glycosylation of the IgG1 Fc domain is required for FcγR binding, though it is unclear why. Structures of the FcγR:Fc complex fail to explain this because the FcγR polypeptide does not bind the N-glycan. Here we identify a link between motion of the N-glycan and Fc:FcγRIIIa affinity that explains the N-glycan requirement. Fc F241 and F243 mutations decreased ...[more]

PMID: 25199692

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Project description:Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure-activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [(13)C,(15)N]-N-acetylglucosamine (GlcNAc). UDP-[(13)C,(15)N]GlcNAc was synthesized enzymatically in a one-pot reaction from [(13)C]glucose and [(15)N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [(13)C,(15)N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

Dataset Information

Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding.

Publications

Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding.

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