Revisiting the Role of Glycosylation in the Structure of Human IgG Fc

Abstract

Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an “open” Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Å resolution and observed significant disorder in the C′E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the CH2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between CH2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (Rg) by small angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger Rg than glycosylated Fc, suggesting a more open CH2 orientation under these conditions. Moreover, the Rg of aglycosylated Fc was reduced by mutations at the CH2-CH3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.

General mechanism for modulating immunoglobulin effector function

“…The CD spectrum of neuraminidase-treated and therefore asialylated human IgG1 Fc (NAse Fc, G2F glycoform) yields the classical spectral pattern associated with β-sheet structure with a minimum at 216 nm and a peak near 203 nm. Deglycosylation of the Fc induces dramatic shifts in the CD spectrum consistent with the structural changes observed in crystal structures of aglycosyl Fc (14,15). Upon sialylation (A2F form), however, we observe a small shift in the spectra for sFc (Fig.…”

“…Thus, the greater solvent-accessible surface area associated with sFc appears to increase hydrophobic surface area as well. Although it is known that the glycan at Asn-297 is required to maintain the quaternary structure of the Cγ2 dimer (14,21), the results shown here indicate that effect of sialylation differs from deglycosylation on Fc structure and stability. Because GnHCl denaturation and ANS binding give comparable results for NAse or deglycosylated Fc (Fig.…”

High-resolution structures of the IgM Fc domains reveal principles of its hexamer formation

“…S4). The increased dynamics of this loop had been already observed in the corresponding IgG Cγ2 domain in the absence of glycosylation in the DE loop (16), suggesting that the DE loop in Cμ3C414S may be stabilized by glycosylation of Asn402. Chemical shift perturbations (CSP) of Cμ3C414S and Cμ3C414S-Cμ4 domain constructs (Fig.…”

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

“…A previous report also describes the effect of two amino acid substitutions (E382V/M428I) that result in high-affinity binding to hFcgRI in the C H 3 domain of an aglycosylated hIgG1 molecule (64). Interestingly, crystallography and small-angle x-ray scattering studies of this Fc fragment show that it has a more closed C H 2-C H 2 conformation than does a similarly aglycosylated WT fragment (65). A recent study (built on crystallization, model building, and simulation of Fc fragments) highlights that the Fc is flexible and can adapt a range of different conformations that are distinct from those observed in FcgR complexes (66).…”