Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire -linked glycans, a process conditional on the introduction of consensus amino acid motifs (-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

The biological and clinical relevance of glycosylation is becoming increasingly recognized, leading to a growing interest in large-scale clinical and population-based studies. In the past few years, several methods for high-throughput analysis of glycans have been developed, but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. In this study, we compared liquid chromatography, capillary gel electrophoresis, and two MS methods for quantitative profiling of N-glycosylation of IgG in the same data set of 1201 individuals. To evaluate the accuracy of the four methods we then performed analysis of association with genetic polymorphisms and age. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and multiplexed capillary gel electrophoresis, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should inform the selection of the most appropriate method in future studies.