Hidden Asymmetric IgG Glycosylation Found in All Humans Drives Dengue Severity

IgG antibodies carry a conserved N-linked glycan at Asn297 on each chain of their homodimeric Fc region, and these glycans powerfully regulate immune effector functions—including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis—by modulating binding to Fc gamma receptors. Decades of research have catalogued how glycan composition (presence or absence of fucose, galactose, sialic acid) correlates with disease severity in conditions ranging from rheumatoid arthritis to COVID-19 and dengue. However, nearly all prior studies used glycan-release methods that strip glycans from the antibody, destroying information about which glycan occupies which Fc protomer arm.

To address this gap, the authors developed WIgGWAM (Whole Immunoglobulin Glycoprofiling With Asymmetric Monitoring). The workflow uses papain digestion to separate Fab from Fc regions, protein A purification of intact homodimeric Fcs, and intact LC/MS analysis in denaturing but non-reducing conditions. This preserves the Fc homodimer and allows assignment of specific glycan pairings to each arm. The method was validated on recombinant monoclonal IgG1s with known glycoforms, then applied to polyclonal IgG1 from human plasma.

Applying WIgGWAM to plasma from healthy adults and COVID-19 patients, the researchers demonstrated that asymmetrically glycosylated IgG1s—where each Fc protomer carries a distinct glycan—are universally present in all individuals tested, not a rare variant. Symmetric glycoforms exist but are a minority. This finding fundamentally challenges the implicit assumption in glycan-release studies that a single glycan composition represents the whole antibody.

Most strikingly, the study revisited the established association between IgG afucosylation and severe dengue disease. Prior glycan-release studies reported elevated afucosylated IgGs in severe and secondary dengue infections. WIgGWAM analysis of dengue patient plasma revealed that the actual driver is asymmetric monofucosylation—IgG1s carrying one fucosylated and one afucosylated Fc glycan—rather than symmetric afucosylation (both Fc arms lacking fucose). Fully afucosylated symmetric IgG glycoforms were not significantly elevated. To explore the functional consequence, the team engineered recombinant monofucosylated IgG1 antibodies. These monofucosylated IgG1s bound FcγRIIIA with the same high affinity as fully afucosylated IgG1s in vitro, and induced comparable antibody-mediated effector functions in vivo in mouse models, suggesting that the afucosylated arm dominates the interaction with the asymmetrically positioned FcγRIII binding site on the Fc.

These findings reframe how IgG glycans regulate immunity and disease. The universality of asymmetric glycosylation means that quantifying glycans without their spatial context misrepresents the true functional units circulating in the blood. The dengue findings suggest that future biomarker studies and therapeutic strategies targeting IgG glycosylation must account for monofucosylated asymmetric species. Engineering monofucosylated antibodies—which may be simpler to produce than fully afucosylated antibodies—could yield a new class of enhanced therapeutic IgGs.