Impact of Fc N-glycan sialylation on IgG structure

Abstract

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.

Keywords: Antibody; IgG1; IgG2; N-glycan; conformation; hydrogen/deuterium exchange; limited proteolysis; mass spectrometry; sialic acid.

Figure 1.

Deuterium uptake curves of glycopeptides (YVDGVEVHNAKTKPREEQYN*STYRVVSVL) containing different glycoforms in human-derived (a) and CHO-expressed (b) IgG1. Data is not corrected for deuterium back exchange. Sialylated glycopeptides are found to have faster H/D exchange rate than other complex glycans in CHO-expressed IgG1, but not human-derived IgG1, indicating that sialylated glycans destabilize the C_H2 domain in CHO-expressed IgG1, but not human-derived IgG1.

Figure 2.

HILIC profiles (SIC of each glycoform) of sialylated N-glycans released from human myeloma and CHO-expressed IgG1 and IgG2, compared to the profiles of α2,3- and α2,6-sialylated glycan libraries. The vertical axes represent the relative signal intensities of each SIC. Panels a and b are profiles of α2,3- and α2,6-sialylated glycan libraries, respectively, panels c and d are profiles of human-derived and CHO-expressed IgG1, respectively, and panels e and f are profiles of human-derived and CHO-expressed IgG2, respectively.

Figure 3.

Reversed-phase profile (SIC of each glycoform) of sialylated glycopeptides released after 24-h tryptic digestion of the human-derived and CHO-expressed IgG1 and IgG2 under a native-like condition. The vertical axes represent the relative signal intensities of each SIC.

Figure 4.

Reversed-phase profile of sialylated glycopeptides after a 3-h tryptic digestion of human-derived IgG1under a native-like condition (a) and followed by sialidase α-(2–3) treatment (b). Disappearance of the minor peaks (A2S1G0F-d, A2S1G1F-d and A2S2F) indicates that all of these minor peaks contain α2,3-linked sialic acid. The vertical axes represent the relative signal intensities of each SIC.

Figure 5.

Reversed-phase profiles of sialylated glycopeptides after a 24-h tryptic digestion of human myeloma IgG1under a native-like condition (a), followed by collection of fractions containing the disialylated glycopeptides (b), then followed by sialidase α-(2–3) treatment (c). Disappearance of A2S2F-b and appearance of A2S1G1F-a indicates that A2S2F-b has α2,3-sialic acid on the 6-arm and α2,6-sialic acid on the 3-arm. The vertical axes represent the relative signal intensities of each SIC.

Figure 6.

Glycan profiles of released glycopeptides (EEQYNSTYR for IgG1 and EEQFNSTFR for IgG2) after tryptic digestion of human-derived and CHO-expressed IgGs for varying lengths of time under a native-like condition. Glycoforms that increase or do not change with time are shown in blue lines and glycoforms that decrease with time are shown in red lines.

Figure 7.

Crystal structure of the α2,6-disialylated glycan in the C_H2 domain (PDB id: 4BYH). The 3-arm is pointing inward, and the 6-arm is pointing outward. Sialic acid on the 3-arm is not observable because of its flexibility. Galactose (yellow) carbon positions are labeled. While sialic acid (purple) linked to the 6-position are exposed to the solvent, linking to the 3-position will likely cause steric effect as it points toward the protein backbone.