Tamara Pavić

Alternative glycosylation modulates function of IgG and other proteins — Implications on evolution and disease

BACKGROUND Nearly all membrane and secreted proteins, as well as numerous intracellular proteins are glycosylated. However, contrary to proteins which are defined by their individual genetic templates, glycans are encoded in a complex dynamic network of hundreds of genes which participate in the complex biosynthetic pathway of protein glycosylation. SCOPE OF REVIEW This review summarizes present knowledge about the importance of alternative glycosylation of IgG and other proteins. MAJOR CONCLUSIONS Numerous proteins depend on correct glycosylation for proper function. Very good example for this is the alternative glycosylation of IgG whose effector functions can be completely changed by the addition or removal of a single monosaccharide residue from its glycans. GENERAL SIGNIFICANCE The change in the structure of a protein requires mutations in DNA and subsequent selection in the next generation, while even slight alterations in activity or intracellular localization of one or more biosynthetic enzymes are sufficient for the creation of novel glycan structures, which can then perform new functions. Glycome composition varies significantly between individuals, which makes them slightly or even significantly different in their ability to execute specific molecular pathways with numerous implications for development and progression of various diseases. This article is part of a Special Issue entitled Glycoproteomics.

Association of systemic lupus erythematosus with decreased immunosuppressive potential of the IgG glycome.

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.

Systemic lupus erythematosus associates with the decreased immunosuppressive potential of the IgG glycome

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.

Glycosylation Profile of IgG in Moderate Kidney Dysfunction

Glycans constitute the most abundant and diverse form of the post-translational modifications, and animal studies have suggested the involvement of IgG glycosylation in mechanisms of renal damage. Here, we explored the associations between IgG glycans and renal function in 3274 individuals from the TwinsUK registry. We analyzed the correlation between renal function measured as eGFR and 76 N-glycan traits using linear regressions adjusted for covariates and multiple testing in the larger population. We replicated our results in 31 monozygotic twin pairs discordant for renal function. Results from both analyses were then meta-analyzed. Fourteen glycan traits were associated with renal function in the discovery sample (P<6.5×10(-4)) and remained significant after validation. Those glycan traits belong to three main glycosylation features: galactosylation, sialylation, and level of bisecting N-acetylglucosamine of the IgG glycans. These results show the role of IgG glycosylation in kidney function and provide novel insight into the pathophysiology of CKD and potential diagnostic and therapeutic targets.

Effects of statins on the immunoglobulin G glycome

BACKGROUND Statins are among the most widely prescribed medications worldwide and usually many individuals involved in clinical and population studies are on statin therapy. Immunoglobulin G (IgG) glycosylation has been associated with numerous cardiometabolic risk factors. METHODS The aim of this study was to investigate the possible association of statin use with N-glycosylation of IgG. The association was analyzed in two large population cohorts (TwinsUK and KORA) using hydrophilic interaction liquid chromatography (HILIC-UPLC) in the TwinsUK cohort and reverse phase liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) in the KORA cohort. Afterwards we investigated the same association for only one statin (rosuvastatin) in a subset of individuals from the randomized double-blind placebo-controlled JUPITER study using LC-ESI-MS for IgG glycome and HILIC-UPLC for total plasma N-glycome. RESULTS In the TwinsUK population, the use of statins was associated with higher levels of core-fucosylated biantennary glycan structure with bisecting N-acetylglucosamine (FA2B) and lower levels of core-fucosylated biantennary digalactosylated monosialylated glycan structure (FA2G2S1). The association between statin use and FA2B was replicated in the KORA cohort. In the JUPITER trial we found no statistically significant differences between the randomly allocated placebo and rosuvastatin groups. CONCLUSIONS In the TwinsUK and KORA cohorts, statin use was associated with a small increase of pro-inflammatory IgG glycan, although this finding was not confirmed in a subset of participants from the JUPITER trial. GENERAL SIGNIFICANCE Even if the association between IgG N-glycome and statins exists, it is not large enough to pose a problem for glycomic studies.

The Association Between Low Back Pain and Composition of IgG Glycome

Low back pain (LBP) is a common debilitating condition which aetiology and pathogenesis are poorly understood. We carried out a first so far analysis of associations between LBP and plasma IgG N-glycome in a sample of 4511 twins from TwinsUK database assessed for LBP, lumbar disc degeneration (LDD) as its possible cause, and IgG-glycan levels. Using weighted correlation network analysis, we established a correlation between LBP and glycan modules featured by glycans that either promote or block antibody-dependent cell-mediated cytotoxicity (ADCC). The levels of four glycan traits representing two of those modules were statistically significantly different in monozygotic twins discordant for LBP. Also, the trend to higher prevalence of systemic inflammatory disorders was shown for twins with low level of fucosylated glycans and high level of non-fucosylated glycans. Core fucosylation of IgG is a “safety switch” reducing ADCC, thus our results suggest the involvement of ADCC and associated inflammation in pathogenesis of LBP. No correlation between LDD scores and glycans was found assuming that the inflammation may not be a part of LDD. These data provide a new insight into understanding the complex pathophysiology of LBP and suggest glycan levels as a possible biomarker for inflammation-related subtypes of LBP.

IgG glycosylation and DNA methylation are interconnected with smoking

BACKGROUND Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic. METHODS With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed. RESULTS The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites. CONCLUSION Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures. GENERAL SIGNIFICANCE An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

Defining the genetic control of human blood plasma N-glycome using genome-wide association study

Glycosylation is a common post-translational modification of proteins. It is known, that glycans are directly involved in the pathophysiology of every major disease. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here, we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people. We discovered and replicated twelve loci. This allowed us to demonstrate a clear overlap in genetic control between total plasma and IgG glycosylation. Majority of loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3, and MGAT5). We, however, also found loci that are likely to reflect other, more complex, aspects of plasma glycosylation process. Functional genomic annotation suggested the role of DERL3, which potentially highlights the role of glycoprotein degradation pathway, and such transcription factor as IKZF1.

Comparison of 2-Aminobenzamide, Procainamide and RapiFluor-MS as Derivatizing Agents for High-Throughput HILIC-UPLC-FLR-MS N-glycan Analysis

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.

Correction to: Increased plasma N-glycome complexity is associated with higher risk of type 2 diabetes

The authors regret that Nish Chaturvedi’s name was spelt incorrectly in the author list. The details given in this correction are correct.