The Structural Role of Antibody N-Glycosylation in Receptor Interactions

Abstract

Asparagine(N)297-linked glycosylation of IgG Fc is required for binding to FcγRIIa, IIb and IIIa though it is unclear how it contributes. We found the quaternary structure of glycosylated Fc was indistinguishable from aglycosylated Fc indicating 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 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 IgG2-4, IgD, IgE and IgM.

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function

“…These changes correlated with differences in crosspeak positions observed with solution nuclear magnetic resonance (NMR) spectroscopy and these differences reflected changes in secondary structure for one IgG1 Fc loop (39). We applied a similar approach and incorporated selective backbone 15 N labels into Y, K and F residues to probe whether Nglycan composition affected 1 H-15 N heteronuclear single quantum coherence (HSQC) spectra of srCD16a.…”

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

“…16 This approach has limitations, including mutagenesis in the C H 3 domains, inability to use a single cell line for production, and, most notably, the final product does not have the conserved N-297 linked glycans in the C H 2 domains that are believed to be important to maintain the structure and function of antibodies. [17][18][19][20][21] A computational design strategy has been applied to express the IgG-Bs in a single mammalian cell by identifying complementary point mutations at the heavy and light chain interfaces. 22 When expressed using the same mammalian cell, the heavy and light chains assemble into IgG-Bs based on their complementary mutations.…”

“…The iMab design offers a unique engineering solution to overcome some of the limitations of the described previously IgG-Bs, [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] such as the potential formation of half-antibodies and homodimers, which can be difficult to eliminate and require complex multi-step purification methods.…”

EndoSd: An IgG Glycan Hydrolyzing Enzyme in Streptococcus Dysgalactiae Subspecies Dysgalactiae

“…The CH2 domain in the Fc region of IgG contains an N-linked glycan attached to asparagine 297 [4]. The stability of Fc region of IgG and interaction with Fc receptors (FcRs) is dependent on the N-linked glycan [5]. The N-linked glycan preorganizes the FcγRIIIa interface for optimal binding affinity, so the glycan plays an essential role in binding of IgG to FcRs [5][6][7].…”

“…The stability of Fc region of IgG and interaction with Fc receptors (FcRs) is dependent on the N-linked glycan [5]. The N-linked glycan preorganizes the FcγRIIIa interface for optimal binding affinity, so the glycan plays an essential role in binding of IgG to FcRs [5][6][7]. Absence of the Fc-glycan in the CH2 domain of IgG diminishes binding to FcRs on leukocytes and reduces complement activation [8].…”