The distinctive structural isomers of IgG Fc-glycans are subclass-specific

Immunoglobulins G (IgGs), are dominant immunoglobulins in the humoral immune response and exhibit dual functionalities: the Fab-domains target antigens, whereas the Fc-domains modulate immune activities through interactions with receptors. In human serum, IgGs are classified into four subclasses, IgG1, IgG2, IgG3 and IgG4, each characterized by unique features in their sequences, disulphide bridges and glycosylation signatures. Fc N-glycosylation plays a key role in modulating the functions of IgGs. Taking IgG1 as an example, changes in the glycans may alter action from inhibitory/anti-inflammatory to activating the complement system and promoting antibody-dependent cellular cytotoxicity.

The functionality of protein glycosylation depends heavily on the precise nature of the linkages and branching between monosaccharides, as well as their specific positions within proteins. Glycosylation research typically involves the analysis of released glycans or glycopeptides. Structural information of glycans is mostly accessible at the released glycan level, yet, the lack of a peptide prevents knowing the proteins of origin (e.g., different IgG subclasses) or the specific glycosylation sites (e.g., the Fc or Fab regions). Glycopeptide analysis, on the other hand, does allow site-specificity, but it greatly underestimates the complexity of the involved glycan structures as it is primarily performed at the compositional level. Method development into studying glycopeptide structure is urgently needed, the chromatographic dimension of LC-MS/MS being a promising direction.

Here, we developed a high-resolution nano-HILIC-LC-MS/MS method capable of quantifying IgG glycan structures at the glycopeptide level. Using this methodology, we demonstrated that IgG subclasses carry differential yet defined structural glycan characteristics, in both recombinant and endogenous IgGs. For instance, our analyses revealed distinct structural glycosylation signatures for each IgG subclass, e.g., IgG1 and IgG3 showing predominant galactosylation of the 6-branched antenna, IgG2 of the 3-branched antenna, and IgG4 a balance of the two. These and other subclass-specific glycostructural elements proved observable in recombinant IgG and in human plasma, in which inter-individual variability and temporal stability could be demonstrated. The here-described onset of structural glycoproteomics is expected to fundamentally alter the way in which we study IgG, opening up a new layer of functional investigation and biomarker development.