NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic

Abstract

The N-glycan at Asn297 of the immunoglobulin G Fc fragment modulates cellular responses of the adaptive immune system. However, the underlying mechanism remains undefined, as existing structural data suggest the glycan does not directly engage cell surface receptors. Here we characterize the dynamics of the glycan termini using solution NMR spectroscopy. Contrary to previous conclusions based on X-ray crystallography and limited NMR data, our spin relaxation studies indicate that the termini of both glycan branches are highly dynamic and experience considerable motion in addition to tumbling of the Fc molecule. Relaxation dispersion and temperature-dependent chemical shift perturbations demonstrate exchange of the α1-6Man-linked branch between a protein-bound and a previously unobserved unbound state, suggesting the glycan samples conformational states that can be accessed by glycan-modifying enzymes and possibly glycan recognition domains. These findings suggest a role for Fc-glycan dynamics in Fc-receptor interactions and enzymatic glycan remodeling.

Figure 1. Immunoglobulin G contains an N-linked glycan at Asn297

(a) The domain organization of IgG1showing the antigen-binding Fab fragments, the Fc fragment, and the N-glycan. (b) The Fc glycan as remodeled in this work is predominantly a galactose-terminated complex-type, biantennary, fucosylated carbohydrate. Open circles indicate galactose, filled squares N-acetylglucosamine, grey circles mannose, and open triangles fucose residues.

Figure 2. A 2D ¹³C-¹H HMQC correlation spectrum of ¹³C-Galactose labeled IgG1 and IgG1 Fab fragment

(a) IgG1 and (b) the IgG1 Fab fragment at 50°C. Vertical grey lines indicate resonances at the same ¹³C and ¹H frequencies in both spectra.

Figure 3. 2D ¹³C-HMQC spectra and assignments of ¹³C-Galactose labeled IgG Fc

(a) uniformly ¹³C-Galactose labeled Fc fragment and (b) ¹³C₂-Galactose labeled Fc fragment. The extracted lines show ¹H linewidth measurements of the H₂-C₂ crosspeaks extracted from this spectrum. (c) IgG Fc fragment with the α1–6Man-linked Galactose enriched with ¹³C-Galactose. Filled (hollow) arrows denote a residue on the α1–3Manlinked (α1–6Man-linked) branch of the Fc-conjugated biantennary glycan. Anomeric peaks were overlapped with the residual HOD signal but were observed at lower temperatures.

Figure 4. ¹³C spin relaxation measurements of Galactose resonances

(a) R₁ and (b) R₂ measurements of ¹³C₆. (c) R₁ and (d) R_1ρ measurements of ¹³C₂. The data are fit with an equation describing a single exponential decay; using the rates shown in Table 1. Where the rates are significantly different, a solid (dashed) line denotes the α1–3Man-linked (α1–6Man-linked) fits. R_1ρ measurements were obtained at an applied field strength of 5000Hz.

Figure 5. Relaxation dispersion and temperature-dependent chemical shift measurements show evidence of two states

Relaxation dispersion experiments (a reveal motion on a µs timescale for the α1–6Man-linked branch but not the α1–3Man-linked branch. When fitted to a two state model and data collected at three different magnetic fields, an exchange rate (k_EX) of 5300 ± 1700 s⁻¹ was estimated by fitting equation (1). (b) The chemical shift of the α1–6Man-linked ¹³C₂ resonance approaches a saturation point at high temperature and permits the estimation of chemical shift values for each of two states using Eq (3). Chemical shift asymptotes for the high temperature state A and low temperature state B are shown with dashed lines.

Figure 6. Models for Fc glycan dynamics and accessibility showing exposed glycan conformations are possible

The terminal galactose residues of the glycan are labeled as “α1–3”Man-linked or “α1–6”Man-linked. These hypothetical models show accessible locations of the glycans in states A and B based on X-ray structures of the Fc fragment (pdb-1l6x). State B is characterized by the α1–6Man-linked branch coordinated by the surface of the polypeptide, and both branches of state A are highly dynamic.