Mouse IgG2c Fc loop residues promote greater receptor-binding affinity than mouse IgG2b or human IgG1

Abstract

The structures of non-human antibodies are largely unstudied despite the potential for the identification of alternative structural motifs and physical properties that will benefit a basic understanding of protein and immune system evolution as well as highlight unexplored motifs to improve therapeutic monoclonal antibody. Here we probe the structure and receptor-binding properties of the mouse IgG2c crystallizable fragment (Fc) to compare to mouse IgG2b and human IgG1 Fcs. Models of mIgG2c Fc determined by x-ray crystallography with a complex-type biantennary (to 2.05 Å) or a truncated (1)GlcNAc asparagine-linked (N)-glycan attached (to 2.04 Å) show differences in key regions related to mouse Fc γ receptor IV (mFcγRIV) binding. Mouse IgG2c forms different non-bonded interactions between the BC, DE and FG loops than the highly-conserved mIgG2b and binds to FcγRIV with 4.7-fold greater affinity in the complex-type glycoform. Secondary structural elements surrounding the Asn297 site of glycosylation form longer beta strands in the truncated mIgG2c Fc glycoform when compared to mIgG2c with the complex-type N-glycan. Solution NMR spectroscopy of the N-linked (1)GlcNAc residues show differences between mIgG2b, 2c and hIgG1 Fc that correlate to receptor binding affinity. Mutations targeting differences in mIgG2 DE and FG loops decreased affinity of mIgG2c for FcγRIV and increased affinity of mIgG2b. Changes in NMR spectra of the mutant Fc proteins mirrored these changes in affinity. Our studies identified structural and functional differences in highly conserved molecules that were not predicted from primary sequence comparison.

Enhanced Immune Responses and Protective Immunity to Zika Virus Induced by a DNA Vaccine Encoding a Chimeric NS1 Fused With Type 1 Herpes Virus gD Protein

“…Since these antibodies are capable to recognize membrane-associated NS1 dimers in ZIKV-infected cells, we expect that, under in vivo conditions, this would favor the clearance of infected cells. The presence of higher IgG2c levels in pgDNS1-ZIKV immunized AB6 mice also supports this hypothesis, since IgG2c antibodies are known to strongly bind to ADCC-mediator FcyRIV (67,68). Thus, the enhanced immunity observed in this group may be related to the role of gD on the modulation of the induced antigen-specific responses.…”

From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses

“…It has also been shown that C H 2 loop motion and relative domain orientation influence the Fc−FcγR interaction in human and mice. 16,24 Interestingly, although the macaque IgG Fcs are very similar in their crystallographic structures and MmIgG1 shows the most structural similarity to HsIgG3, solution NMR spectroscopy detected the same or greater motion of the N 297 glycan among the MmIgG Fcs as observed for HsIgG1 Fc. In addition, MmIgG1 spectra exhibited GlcNAc 1 H1-C1 peak features most similar to that of HsIgG1.…”

“…23 Furthermore, the positions of comparable peaks from mouse IgG2b and 2c Fc also correlate with FcγRIV binding affinity. 24 Thus, this H 1 -C 1 correlation represents a well studied and valuable reporter of IgG Fc structure. The GlcNAc 1 H 1 -C 1 correlations appeared at comparable resonance frequencies for all IgG Fcs tested (Figure 5(a)).…”

The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding

“…The same analytical approach was applied to mouse IgG2b Fc and IgG2c Fc. Unlike human Fc, the mouse proteins showed only one peak for the Asn-linked N-acetylglucosamine residue; however, the peak from IgG2c Fc was found further to the right and mIgG2c bound mouse Fc γ receptor IV with ~5-fold greater affinity that mIgG2b Fc, consistent with the stabilized conformation observed in hIgG1 Fc (Falconer & Barb, 2018). …”