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

Abstract

The Old World monkey, Rhesus macaque (Macaca mulatta, Mm), is frequently used as a primate model organism in the study of human disease and to test new vaccines/antibody treatments despite diverging before chimpanzees and orangutans. Mm and humans share 93% genome identity with substantial differences in the genes of the adaptive immune system that lead to different functional IgG subclass characteristics, Fcγ receptors expressed on innate immune cells, and biological interactions. These differences put limitations on Mm use as a primary animal model in the study of human disease and to test new vaccines/antibody treatments. Here, we comprehensively analyzed molecular properties of the Fc domain of the four IgG subclasses of Rhesus macaque to describe potential mechanisms for their interactions with effector cell Fc receptors. Our studies revealed less diversity in the overall structure among the Mm IgG Fc, with MmIgG1 Fc being the most structurally like human IgG3, although its C_H2 loops and N²⁹⁷ glycan mobility are comparable to human IgG1. Furthermore, the Fcs of Mm IgG3 and 4 lack the structural properties typical for their human orthologues that determine IgG3's reduced interaction with the neonatal receptor and IgG4's ability for Fab-arm exchange and its weaker Fcγ receptor interactions. Taken together, our data indicate that MmIgG1-4 are less structurally divergent than the human IgGs, with only MmIgG1 matching the molecular properties of human IgG1 and 3, the most active IgGs in terms of Fcγ receptor binding and Fc-mediated functions. PDB accession numbers for deposited structures are 6D4E, 6D4I, 6D4M, and 6D4N for MmIgG1 Fc, MmIgG2 Fc, MmIgG3 Fc, and MmIgG4 Fc, respectively.

Keywords: Fc; IgG subclasses; Macaca mulatta; Rhesus macaque; crystallizable fragment.

Figure 1.

Crystal structures of the Rhesus macaque IgG1-4 Fc. (a) The overall structures are shown in a ribbon diagram with the two heavy chains (C_H2-C_H3 domains) in lighter and darker shades of green (MmIgG1), pink (MmIgG2), blue (MmIgG3) and yellow (MmIgG4). The sugars attached to N²⁹⁷ are shown as spheres colored by atom type (backbone color for carbon; red for oxygen and blue for nitrogen). The distances between Cα carbons of P²³⁸ are shown to indicate the differences in the distances between C_H2 domains. (b) The structures of MmIgG1-4 Fcs were superimposed based on C_H3-C_H3 homodimer to show differences in the conformation and distances of C_H2 domains in the Fc dimer. A 45° view shows the conformation of C’E, BC, and FG loops among C_H2 domains of the Fcs. (c) Sequence alignment of the four subtypes of MmIgG Fc. The sequence identity among the four sequences is 86%. Residues of MmIgG2-4 different than MmIgG1 sequence are shaded in pink. The secondary elements as determined by the structures are shown above the sequence with arrows for β–strands, cylinders for α-helix and solid lines for random coil. Residue N²⁹⁷ is indicated with a red star and the C’E, BC, and FG loops are in boxes. (d) C_H2−C_H3 interface. Residues contributing to the interface through salt bridges/hydrogen bonds and residues of the hydrophobic “ball-in socket” joint are shown as sticks. Distances for hydrogen bonds/electrostatic interactions are as shown.

Figure 2.

Comparison of the overall structures of the Rhesus macaque and human IgG1-4 Fc. (a) Average RMSD values for main chain atoms for pairwise comparisons of C_H2, C_H3, C_H2-C_H3 monomers and C_H2-C_H3 dimers (Fc domain). The structures of the Fcs of human IgG1-4 used in the alignments include: IgG1, PDB codes: 3AVE, 4DZ8, 4W4N, 1H3Y and 1H3V; IgG2, PDB codes: 4HAF, 4HAG; IgG3, PDB code: 5W38 and IgG4, PDB codes: 4C54, 4C55, 5LG1. (b) Structural alignment of C_H2, C_H3, C_H2-C_H3 monomers, and C_H2-C_H3 dimers. MmFcs are colored in green for IgG1, pink for IgG2, blue for IgG3 and yellow for IgG4. Human Fcs are colored in grey. C_H2-C_H3 dimers are aligned by superimposing the C_H3 domains.

Figure 3.

Structural comparisons of the Rhesus macaque and human Fcs. (a) Pairwise comparisons of the overall structures shown in a ribbon diagram with the two heavy chains (C_H2-C_H3 domains) in lighter and darker shades of green (MmIgG1), pink (MmIgG2), blue (MmIgG3) and yellow (MmIgG4) overlaid on their human counterpart in grey. The sugars attached to N²⁹⁷ are shown as sticks and side chains for residues that differ between macaque and human shown as balls and sticks colored by atom type (backbone color for carbon; red for oxygen and blue for nitrogen). Residues in the BC, C’E, and FG loops known in human to contribute to the Fcγ receptor binding are highlighted by color-matched circles. The same color scheme is used in remaining panels. (b) The conformation of C’E, BC, and FG loops among C_H2 domains of the Fcs. (c) Pairwise sequence alignment of the four subtypes of macaque and human IgG Fc. Residues that are different between macaque and human are shaded in pink. The C’E, BC, and FG loops are in boxes. Residue N²⁹⁷ is indicated with a red star and residues at positions 405, 410 and 435 are indicated by a red arrow.

Figure 4.

Differences in spectral quality in ¹H-¹³C HSQC spectra of [¹³CU-glycan]-Fcs collected at 18.8 T and 50ºC. These spectra were processed with only a sine-squared line-broadening function in the direct dimension. The vertical arrow highlights the peak which corresponds to the GlcNAc¹ H⁶-C¹ correlation. Peaks within the dashed boxes are used for comparisons of spectra in the main text.

Figure 5.

Differences in the anomeric ¹H^1-13C¹ correlation in the N²⁹⁷-linked GlcNAc¹ residue (a) at 18.8 T and 50ºC and (b) at 14.1 T and 50ºC. These spectra were processed with a combination of sine-squared and 20 Hz exponential multiplier line-broadening functions in the direct dimension.

Figure 6.

Analysis of the N-glycan composition of the Fcs of MmIgG1-4 expressed in HEK293F cells and present in RM sera. (a) N-glycan composition of the Fcs of MmIgG1-4 expressed in HEK293F is shown with NeuAc = N-acetylneuraminic acid; Gal = galactose. (a) left panel shows the percentage of complex-type N-glycans. The upper right panel shows the percent fucosylated, the lower left shows the percentage with one or two sialic acid residues and the lower right indicates galactosylation. (b) and (c) Composition and configuration of the Fc N-glycan of MmIgG1-4 present in RM sera. Comparable results from a different animal are shown in Figure S3. Fc N-glycans identified by LC-ESI-MS/MS are ranked according to spectral counts (panel B). Incidence of each N-glycan modification observed. NeuGc = N-glycolylneuraminic acid; Gal = galactose (panel C).