Fine structural details of glycans attached to the conserved N-glycosylation site significantly not only affect function of individual immunoglobulin G (IgG) molecules but also mediate inflammation at the systemic level. By analyzing IgG glycosylation in 5,117 individuals from four European populations, we have revealed very complex patterns of changes in IgG glycosylation with age. Several IgG glycans (including FA2B, FA2G2, and FA2BG2) changed considerably with age and the combination of these three glycans can explain up to 58% of variance in chronological age, significantly more than other markers of biological age like telomere lengths. The remaining variance in these glycans strongly correlated with physiological parameters associated with biological age. Thus, IgG glycosylation appears to be closely linked with both chronological and biological ages. Considering the important role of IgG glycans in inflammation, and because the observed changes with age promote inflammation, changes in IgG glycosylation also seem to represent a factor contributing to aging.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27×10⁻⁹) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer. After analysing glycans attached to human immunoglobulin G in 4,095 individuals, we performed the first genome-wide association study (GWAS) of the glycome of an individual protein. Nine genetic loci were found to associate with glycans with genome-wide significance. Of these, four were enzymes that directly participate in IgG glycosylation, thus the observed associations were biologically founded. The remaining five genetic loci were not previously implicated in protein glycosylation, but the most of them have been reported to be relevant for autoimmune and inflammatory conditions and/or haematological cancers. A particularly interesting gene, IKZF1 was found to be associated with multiple IgG N-glycans. This gene has been implicated in numerous diseases, including systemic lupus erythematosus (SLE). We analysed N-glycans in 101 cases with SLE and 183 matched controls and demonstrated their substantial biomarker potential. Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. Our results may also provide an explanation for opposite effects of some genes in autoimmune diseases and haematological cancer.

Objective: Glycans attached to the Fc portion of IgG are important modulators of IgG effector functions. Interindividual differences in IgG glycome composition are large and they associate strongly with different inflammatory and autoimmune diseases. IKZF1, HLA–DQ2A/B, and BACH2 genetic loci that affect IgG glycome composition show pleiotropy with systemic lupus erythematosus (SLE), indicating a potentially causative role of aberrant IgG glycosylation in SLE. We undertook this large multicenter case–control study to determine whether SLE is associated with altered IgG glycosylation.Methods: Using ultra‐performance liquid chromatography analysis of released glycans, we analyzed the composition of the IgG glycome in 261 SLE patients and 247 matched controls of Latin American Mestizo origin (the discovery cohort) and in 2 independent replication cohorts of different ethnicity (108 SLE patients and 193 controls from Trinidad, and 106 SLE patients and 105 controls from China).Results: Multiple statistically significant differences in IgG glycome composition were observed between patients and controls. The most significant changes included decreased galactosylation and sialylation of IgG (which regulate proinflammatory and antiinflammatory actions of IgG) as well as decreased core fucose and increased bisecting N‐acetylglucosamine (which affect antibody‐dependent cell‐mediated cytotoxicity).Conclusion: The IgG glycome in SLE patients is significantly altered in a way that decreases immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the intensity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in SLE.

Recovery after cardiac surgery is a complex process that has to compensate for both individual variability and extensive tissue damage in the context of systemic inflammation. Protein glycosylation is essential in many steps of the inflammatory cascade, but due to technological limitations the role of individual variation in glycosylation in systemic inflammation has not been addressed until now. We analysed composition of the total plasma and IgG N-glycomes in 107 patients undergoing cardiac surgery. In nearly all individuals plasma N-glycome underwent the same pattern of changes in the first 72[emsp14]h, revealing a general mechanism of glycosylation changes. To the contrary, changes in the IgG glycome were very individualized. Bi-clustering analysis revealed the existence of four distinct patterns of changes. One of them, characterized by a rapid increase in galactosylated glycoforms, was associated with nearly double mortality risk measured by EuroSCORE II. Our results indicate that individual variation in IgG glycosylation changes during acute systemic inflammation associates with increased mortality risk and indicates new avenues for the development of personalized diagnostic and therapeutic approach.

Effector functions of immunoglobulin G (IgG) are regulated by the composition of a glycan moiety, thus affecting activity of the immune system. Aberrant glycosylation of IgG has been observed in many diseases, but little is understood about the underlying mechanisms. We performed a genome-wide association study of IgG N-glycosylation (N = 8090) and, using a data-driven network approach, suggested how associated loci form a functional network. We confirmed in vitro that knockdown of IKZF1 decreases the expression of fucosyltransferase FUT8, resulting in increased levels of fucosylated glycans, and suggest that RUNX1 and RUNX3, together with SMARCB1, regulate expression of glycosyltransferase MGAT3. We also show that variants affecting the expression of genes involved in the regulation of glycoenzymes colocalize with variants affecting risk for inflammatory diseases. This study provides new evidence that variation in key transcription factors coupled with regulatory variation in glycogenes modifies IgG glycosylation and has influence on inflammatory diseases.

Rising awareness of the universal importance of protein N-glycosylation governs the development of further advances in N-glycan analysis. Nowadays it is well known that correct glycosylation is essential for proper protein function, which emanates from its important role in many physiological processes. Furthermore, glycosylation is involved in pathophysiology of multiple common complex diseases. In the vast majority of cases, N-glycosylation profiles are analyzed from enzymatically released glycans, which can be further derivatized in order to enhance the sensitivity of the analysis. Techniques wherein derivatized N-glycans are profiled using hydrophilic interaction chromatography (HILIC) with fluorescence (FLR) and mass spectrometry (MS) detection are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the performance of frequently used labeling compounds −2-aminiobenzamide (2-AB) and procainamide (ProA), and the recently introduced RapiFluor-MS (RF-MS) fluorescent tag. In all experiments N-glycans were released by PNGase F, fluorescently derivatized, purified by HILIC solid phase extraction and profiled using HILIC-UPLC-FLR-MS. We assessed sensitivity, linear range, limit of quantification (LOQ), repeatability and labeling efficiency for all three labels. For this purpose, we employed in-house prepared IgG and a commercially available IgG as a model glycoprotein. All samples were analyzed in triplicates using different amounts of starting material. We also tested the performance of all three labels in a high-throughput setting on 68 different IgG samples, all in duplicates and 22 identical IgG standards. In general, ProA labeled glycans had the highest FLR sensitivity (15-fold and 4-fold higher signal intensities compared to 2-AB and RF-MS respectively) and RF-MS had the highest MS sensitivity (68-fold and 2-fold higher signal intensities compared to 2-AB and ProA, respectively). ProA and RF-MS showed comparable limits of quantification with both FLR and MS detection, whilst 2-AB exhibited the lowest sensitivity. All labeling procedures showed good and comparable repeatability. Furthermore, the results indicated that labeling efficiency was very similar for all three labels. In conclusion, all three labels are a good choice for N-glycan derivatization in high-throughput HILIC-UPLC-FLR-MS N-glycan analysis, although ProA and RF-MS are a better option when higher sensitivity is needed.