The Structural Role of Antibody N-Glycosylation in Receptor Interactions

Abstract

Asparagine(N)297-linked glycosylation of immunoglobulin G (IgG) Fc is required for binding to FcγRIIa, IIb, and IIIa, although it is unclear how it contributes. We found the quaternary structure of glycosylated Fc was indistinguishable from aglycosylated Fc, indicating that N-glycosylation does not maintain relative Fc Cγ2/Cγ3 domain orientation. However, the conformation of the C'E loop, which contains N297, was significantly perturbed in the aglycosylated Fc variant. The conformation of the C'E loop as measured with a range of Fc variants shows a strong correlation with FcγRIIIa affinity. These results indicate that the primary role of the IgG1 Fc N-glycan is to stabilize the C'E loop through intramolecular interactions between carbohydrate and amino acid residues, and preorganize the FcγRIIIa interface for optimal binding affinity. The features that contribute to the capacity of the IgG1 Fc N-glycan to restrict protein conformation and tune binding affinity are conserved in other antibodies including IgG2-IgG4, IgD, IgE, and IgM.

Figure 1

Structure of the IgG1 Fc / FcγRIIIa complex. (A) The IgG1 Fc dimer (blue ribbon) binds to the extracellular domain of FcγRIIIa (orange ribbon) with high nM affinity. A substantial contact interface is formed by the C'E loop of one Fc chain (highlighted in yellow) and the receptor polypeptide. (B) The IgG Fc N-glycan, shown here in a cartoon diagram according to the CfG convention (Varki, 2009), is required for FcγRIIIa binding. Numbers in the cartoon shapes represent the numbering strategy used to describe individual N-glycan residues. (C) The motion of the Fc N-glycan is restricted by contacts with the polypeptide surface, including F241, F243 and D265.

Figure 2

NMR indicates that Fc wt Cγ2 domain orientation, relative to the Cγ3 domain, is nearly identical to that observed by x-ray crystallography and changes little upon removing the N-glycan. (A) Orientations of the Cγ2 domains are overlaid following optimization of the Cγ2 orientation, alignment of the Cγ3 domains (not shown) and centering Cγ2 domains by their centers of mass. The Fc model solved by x-ray crystallography is shown as a blue ribbon. The orientations of Fc wt in solution (red ribbon) and the non-glycosylated variant Fc T299A (green ribbon) are also shown. Comparisons to the observed versus calculated RDCs for Fc wt and Fc T299A are shown in (B) and (C). The Fc N-glycan is not shown.

Figure 3

A ¹H-¹⁵N-HSQC TROSY spectrum reveals that N-glycan removal (with a T299A mutation) perturbs resonances in the C'E loop (Y296 & 300) and along the C' strand (K288 and K290). (A) These four resonances are found on the same structural feature as the N297 glycosylation site. (B) Y296 was not observed in spectra of glycosylated Fc wt. Fc assignments were reported by Kato and coworkers (Yagi et al., 2014).

Figure 4

NMR measurements indicate C'E loop structure and motion is stabilized on the microsecond-millisecond timescale by N297 glycosylation. (A) Differences between peak positions of IgG1 Fc wt and the T299A variant are plotted as described (Farmer et al., 1996). The differences between backbone amide nitrogen relaxation rates from IgG1 Fc wt and the T299A variant measured using (B) R₂ and (C) R¹ experiments are shown. Error bars indicate the precision of measurement for each set of relaxation rates (+/− 1 SD).

Figure 5

The peak corresponding to Y300 is displaced in spectra of Fc variants. (A) ¹H-¹⁵N HSQC-TROSY spectra of [¹⁵N-Y]-Fcs and FcγRIIIa affinity of Fc variants. The curved black arrow shows the direction of chemical shift change of the Y300 peak with decreasing FcγRIIIa affinity. (B) Assignment of the Y300 residue in Fc D265A spectrum was performed by comparison with a spectrum of the Fc Y300F/D265A variant.

Figure 6

The (1)GlcNAc residue experiences multiple chemical environments in Fc wt, but only one state is present in proteolyzed Fc or Fc variants that bind FcγRIIIa weakly (>50 µM). ¹H-¹³C HSQC spectra of ¹³C-labeled Fc variants are drawn to highlight the spectral region corresponding to the anomeric region of the (1)GlcNAc residue. The vertical dashed line corresponds to the ¹H chemical shift of the upfield signal in the Fc wt protein with a complex-type N-glycan. A complete spectrum of the Fc samples is shown in Supplemental Figure 6.

Figure 7

The D265A Fc variant retains significant contact between the polypeptide and N-glycan termini as observed with the (6) and (6') galactose residues. ¹H-¹³C HSQC spectra of (A) IgG1 Fc wt and (B) D265A variant. N-glycans were remodeled to display the G2F glycoform with ¹³C2-galactose. Positions of Fc wt peaks are highlighted by “+”. Resonance frequencies consistent with an exposed and unrestricted N-glycan are labeled “e” (Subedi et al., 2014).

Figure 8

A comprehensive model of Fc conformations. The Fc N-glycan is required to restrict the C'E loop, and state iv represents the dominant conformation of Fc wt that is also predisposed to bind FcγRIIIa. The equilibrium established between states iii and iv can be perturbed by removing the N-glycan or perturbing the N-glycan interface. For complete exposure of the N-glycan, known to occur based on the sensitivity to glycan modifying enzymes that bind carbohydrate motifs consisting of 6+ residues, the Cγ2 reorients to allow for complete dissociation of the N-glycan from the polypeptide surface and the Fc interstitial space.