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Antibodies are known to have an important role in the development of rheumatoid arthritis (RA), one of the most prevalent chronic inflammatory diseases which primarily involves the joints. Most RA patients develop autoantibodies against immunoglobulin G (IgG) and changes in IgG glycosylation have been associated with RA. We undertook this study to determine whether altered IgG glycosylation precedes the disease diagnosis. We studied IgG glycosylation in RA in two prospective cohorts (N = 14,749) by measuring 28 IgG glycan traits in 179 subjects who developed RA within 10-years follow-up and 358 matched controls. Ultra-performance liquid chromatography method based on hydrophilic interactions (HILIC-UPLC) was used to analyse IgG glycans. Future RA diagnosis associated with traits related to lower galactosylation and sialylation of IgG when comparing the cases to the matched controls. In RA cases, these traits did not correlate with the time between being recruited to the study and being diagnosed with RA (median time 4.31 years). The difference in IgG glycosylation was relatively stable and present years before diagnosis. This indicates that long-acting factors affecting IgG glycome composition are among the underlying mechanisms of RA and that decreased galactosylation is a pre-existing risk factor involved in the disease development., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Rationale: One measure of protein glycosylation (GlycA) has been reported to predict higher cardiovascular risk by reflecting inflammatory pathways., Objective: The main objective of this study is to assess the role of a comprehensive panel of IgG glycosylation traits on traditional risk factors for cardiovascular disease and on presence of subclinical atherosclerosis in addition to GlycA., Methods and Results: We measured 76 IgG glycosylation traits in 2970 women (age range, 40-79 years) from the TwinsUK cohort and correlated it to their estimated 10-year atherosclerotic cardiovascular disease risk score and their carotid and femoral plaque measured by ultrasound imaging. Eight IgG glycan traits are associated with the 10-year atherosclerotic cardiovascular disease risk score after adjusting for multiple tests and for individual risk factors-5 with increased risk and 3 with decreased risk. These glycans replicated in 967 women from ORCADES cohort (Orkney Complex Disease Study), and 6 of them were also associated in 845 men. A linear combination of IgG glycans and GlycA is also associated with presence of carotid (odds ratio, 1.55; 95% confidence interval, 1.25-1.93; P =7.5×10 -5 ) and femoral (odds ratio, 1.32; 95% confidence interval, 1.06-1.64; P =0.01) plaque in a subset of women with atherosclerosis data after adjustment for traditional risk factors. One specific glycosylation trait, GP18-the percentage of FA2BG2S1 glycan in total IgG glycans, was negatively correlated with very-low-density lipoprotein and triglyceride levels in serum and with presence of carotid plaque (odds ratio, 0.60; 95% confidence interval, 0.50-0.71; P =5×10 -4 )., Conclusions: We find molecular pathways linking IgG to arterial lesion formation. Glycosylation traits are independently associated with subclinical atherosclerosis. One specific trait related to the sialylated N-glycan is negatively correlated with cardiovascular disease risk, very-low-density lipoprotein and triglyceride serum levels, and presence of carotid plaque., (© 2018 The Authors.)

Immunoglobulin G (IgG) glycosylation is essential for function of the immune system, but the genetic and environmental factors that underlie its inter-individual variability are not well defined. The Collaborative Cross (CC) genetic resource harnesses over 90% of the common genetic variation of the mouse. By analyzing the IgG glycome composition of 95 CC strains, we made several important observations: (i) glycome variation between mouse strains was higher than between individual humans, despite all mice having the same environmental influences; (ii) five genetic loci were found to be associated with murine IgG glycosylation; (iii) variants outside traditional glycosylation site motifs affected glycome variation; (iv) bisecting N-acetylglucosamine (GlcNAc) was produced by several strains although most previous studies have reported the absence of glycans containing the bisecting GlcNAc on murine IgGs; and (v) common laboratory mouse strains are not optimal animal models for studying effects of glycosylation on IgG function.

Background and Aims: Causes of inflammatory bowel diseases are not well understood and the most prominent forms, Crohn's disease (CD) and ulcerative colitis (UC), are sometimes hard to distinguish. Glycosylation of IgG has been associated with CD and UC. IgG Fc-glycosylation affects IgG effector functions. We evaluated changes in IgG Fc-glycosylation associated with UC and CD, as well as with disease characteristics in different patient groups., Methods: We analyzed 3441 plasma samples obtained from 2 independent cohorts of patients with CD (874 patients from Italy and 391 from the United States) or UC (1056 from Italy and 253 from the US and healthy individuals [controls]; 427 in Italy and 440 from the United States). IgG Fc-glycosylation (tryptic glycopeptides) was analyzed by liquid chromatography coupled to mass spectrometry. We analyzed associations between disease status (UC vs controls, CD vs controls, and UC vs CD) and glycopeptide traits, and associations between clinical characteristics and glycopeptide traits, using a logistic regression model with age and sex included as covariates., Results: Patients with CD or UC had lower levels of IgG galactosylation than controls. For example, the odds ratio (OR) for IgG1 galactosylation in patients with CD was 0.59 (95% confidence interval [CI], 0.51-0.69) and for patients with UC was 0.81 (95% CI, 0.71-0.92). Fucosylation of IgG was increased in patients with CD vs controls (for IgG1: OR, 1.27; 95% CI, 1.12-1.44), but decreased in patients with UC vs controls (for IgG23: OR, 0.72; 95% CI, 0.63-0.82). Decreased galactosylation associated with more severe CD or UC, including the need for surgery in patients with UC vs controls (for IgG1: OR, 0.69; 95% CI, 0.54-0.89) and in patients with CD vs controls (for IgG23: OR, 0.78; 95% CI, 0.66-0.91)., Conclusions: In a retrospective analysis of plasma samples from patients with CD or UC, we associated levels of IgG Fc-glycosylation with disease (compared to controls) and its clinical features. These findings could increase our understanding of mechanisms of CD and UC pathogenesis and be used to develop diagnostics or guide treatment., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Immunoglobulin G (IgG), a glycoprotein secreted by plasma B-cells, plays a major role in the human adaptive immune response and are associated with a wide range of diseases. Glycosylation of the Fc binding region of IgGs, responsible for the antibody's effector function, is essential for prompting a proper immune response. This study focuses on the general genetic impact on IgG glycosylation as well as corresponding subclass specificities. To identify genetic loci involved in IgG glycosylation, we performed a genome-wide association study (GWAS) on liquid chromatography electrospray mass spectrometry (LC-ESI-MS)-measured IgG glycopeptides of 1,823 individuals in the Cooperative Health Research in the Augsburg Region (KORA F4) study cohort. In addition, we performed GWAS on subclass-specific ratios of IgG glycans to gain power in identifying genetic factors underlying single enzymatic steps in the glycosylation pathways. We replicated our findings in 1,836 individuals from the Leiden Longevity Study (LLS). We were able to show subclass-specific genetic influences on single IgG glycan structures. The replicated results indicate that, in addition to genes encoding for glycosyltransferases (i.e., ST6GAL1, B4GALT1, FUT8 , and MGAT3 ), other genetic loci have strong influences on the IgG glycosylation patterns. A novel locus on chromosome 1, harboring RUNX3 , which encodes for a transcription factor of the runt domain-containing family, is associated with decreased galactosylation. Interestingly, members of the RUNX family are cross-regulated, and RUNX3 is involved in both IgA class switching and B-cell maturation as well as T-cell differentiation and apoptosis. Besides the involvement of glycosyltransferases in IgG glycosylation, we suggest that, due to the impact of variants within RUNX3 , potentially mechanisms involved in B-cell activation and T-cell differentiation during the immune response as well as cell migration and invasion involve IgG glycosylation.

Correction to: Nature Communications (2017) 8:1231. doi:10.1038/s41467-017-01525-0.

Objective: Poorer glycemic control in type 1 diabetes may alter N-glycosylation patterns on circulating glycoproteins, and these alterations may be linked with diabetic kidney disease (DKD). We investigated associations between N-glycans and glycemic control and renal function in type 1 diabetes., Research Design and Methods: Using serum samples from 818 adults who were considered to have extreme annual loss in estimated glomerular filtration rate (eGFR; i.e., slope) based on retrospective clinical records, from among 6,127 adults in the Scottish Diabetes Research Network Type 1 Bioresource Study, we measured total and IgG-specific N-glycan profiles. This yielded a relative abundance of 39 total (GP) and 24 IgG (IGP) N-glycans. Linear regression models were used to investigate associations between N-glycan structures and HbA 1c , albumin-to-creatinine ratio (ACR), and eGFR slope. Models were adjusted for age, sex, duration of type 1 diabetes, and total serum IgG., Results: Higher HbA 1c was associated with a lower relative abundance of simple biantennary N-glycans and a higher relative abundance of more complex structures with more branching, galactosylation, and sialylation (GP12, 26, 31, 32, and 34, and IGP19 and 23; all P < 3.79 × 10 -4 ). Similar patterns were seen for ACR and greater mean annual loss of eGFR, which were also associated with fewer of the simpler N-glycans (all P < 3.79 × 10 -4 )., Conclusions: Higher HbA 1c in type 1 diabetes is associated with changes in the serum N-glycome that have elsewhere been shown to regulate the epidermal growth factor receptor and transforming growth factor-β pathways that are implicated in DKD. Furthermore, N-glycans are associated with ACR and eGFR slope. These data suggest that the role of altered N-glycans in DKD warrants further investigation., (© 2017 by the American Diabetes Association.)

Introduction: Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins. Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context. Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no 'gold-standard' approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions.

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Osteoarthritis (OA) is one of the leading musculoskeletal disorders in the adult population. It is associated with cartilage damage triggered by the deterioration of the extracellular matrix tissue. The present study explores the effect of intra-articular injection of autologous microfragmented adipose tissue to host chondrocytes and cartilage proteoglycans in patients with knee OA. A prospective, non-randomized, interventional, single-center, open-label clinical trial was conducted from January 2016 to April 2017. A total of 17 patients were enrolled in the study, and 32 knees with osteoarthritis were assessed. Surgical intervention (lipoaspiration) followed by tissue processing and intra-articular injection of the final microfragmented adipose tissue product into the affected knee(s) was performed in all patients. Patients were assessed for visual analogue scale (VAS), delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) and immunoglobulin G (IgG) glycans at the baseline, three, six and 12 months after the treatment. Magnetic resonance sequence in dGEMRIC due to infiltration of the anionic, negatively charged contrast gadopentetate dimeglumine (Gd-DTPA 2- ) into the cartilage indicated that the contents of cartilage glycosaminoglycans significantly increased in specific areas of the treated knee joint. In addition, dGEMRIC consequently reflected subsequent changes in the mechanical axis of the lower extremities. The results of our study indicate that the use of autologous and microfragmented adipose tissue in patients with knee OA (measured by dGEMRIC MRI) increased glycosaminoglycan (GAG) content in hyaline cartilage, which is in line with observed VAS and clinical results., Competing Interests: The authors declare no conflict of interest.

Joint modeling of a number of phenotypes using multivariate methods has often been neglected in genome-wide association studies and if used, replication has not been sought. Modern omics technologies allow characterization of functional phenomena using a large number of related phenotype measures, which can benefit from such joint analysis. Here, we report a multivariate genome-wide association studies of 23 immunoglobulin G (IgG) N-glycosylation phenotypes. In the discovery cohort, our multi-phenotype method uncovers ten genome-wide significant loci, of which five are novel (IGH, ELL2, HLA-B-C, AZI1, FUT6-FUT3). We convincingly replicate all novel loci via multivariate tests. We show that IgG N-glycosylation loci are strongly enriched for genes expressed in the immune system, in particular antibody-producing cells and B lymphocytes. We empirically demonstrate the efficacy of multivariate methods to discover novel, reproducible pleiotropic effects.Multivariate analysis methods can uncover the relationship between phenotypic measures characterised by modern omic techniques. Here the authors conduct a multivariate GWAS on IgG N-glycosylation phenotypes and identify 5 novel loci enriched in immune system genes.

Next-generation (postgenomic) biomarkers from the nascent field of glycomics now offer fresh vistas for innovation in chronic disease biomarkers and system diagnostics in clinical medicine. Our previous work has shown an association between hypertension and immunoglobulin G (IgG) glycome composition, suggesting that individual variation in N-glycosylation of IgG might contribute to hypertension pathogenesis. The present study examined, for the first time to the best of our knowledge, the IgG N-glycans as potential biomarkers for hypertension in the Kazakh population. The profile of 60 N-glycopeptides of IgG subclass isolated from plasma samples of 150 Kazakh study participants was analyzed by nano ultra-performance liquid chromatography with mass spectrometry. Fourteen IgG subclass-specific Fc N-glycopeptide structures, along with one derived glycosylation trait in subclasses IgG2/3 and IgG4, were found to correlate with systolic blood pressure and/or diastolic blood pressure. For differentiation of hypertension and healthy status in the Kazakh population sample, the receiver operating characteristic curve analysis showed that the performance of the model, including nine IgG N-glycans, was greater than the traditional gender, age, and body mass index based model (p < 0.05). This study indicates that alteration in Fc N-glycopeptide profiles of plasma IgG subclasses is associated with blood pressure status in the Kazakh population. IgG N-glycosylation profiles may serve as potential biomarkers for hypertension in the Kazakhs, thus contributing to move toward personalized medicine. Further studies of postgenomic glycomic biomarkers in cardiovascular and chronic diseases are timely and called for.

Background: Image-guided tumor ablation is a technique whereby needle-like applicators are placed directly into solid tumors under guidance typically with computed tomography or ultrasound. Changes in IgG and IgM antibody glycosylation were studied during ablation-induced immune response to cancer, and the use of glycosylation as a biomarker for diagnosis, prognosis and disease treatment was examined., Methods: Plasma from 27 tumor patients was collected immediately before, after and for 6 months following ablation. IgG and IgM antibodies were isolated by use high-throughput chromatography, and analyzed by hydrophilic liquid chromatography. Thorough identification of glycan structures in each chromatography peak was performed by nano-liquid chromatography electrospray ionization mass spectrometry., Results: Although antibody glycosylation was found to vary with cancer type, discernable patterns of change based on the successful treatment of tumors by ablation were not identified. One patient with renal clear cell carcinoma and poor disease outcome had unexpectedly high amount of oligomannose IgG glycans during the whole period of monitoring. In contrast, IgM antibodies did not follow the same pattern., Conclusions: These findings suggest that glycosylation patterns are indicative of an immune system that is unable to prevent different types of cancer, rather than products of the immunostimulatory response to the ablation of tumor itself. Analyses of the outcome effect suggested that IgG glycosylation and IgM glycosylation are not associated with tumor ablation., General Significance: Present work opens a new way for parallel determination of glycosylation changes of both IgG and IgM antibodies by use of high-throughput methods, and their future use as biomarkers for disease diagnosis and prognosis. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Glycosylation of immunoglobulin G (IgG) is important for its effector functions and was shown to be related to age, sex and disease status of an individual. Adding glycomic information to genome-wide association studies (GWAS) and large clinical trials is enabling insight into the functional relevance of changes in glycosylation, as well as molecular mechanisms behind these changes. Large-scale studies require sensitive, robust and affordable high-throughput methodologies for glycosylation analysis, which are currently available in only a limited number of laboratories., Areas Covered: This review focuses on currently used high-throughput approaches for N-glycosylation analysis of IgG, as well as some recent advances in the areas of deglycosylation, trypsin digestion, labeling, purification, derivatization and automation of current workflows. Relevant literature was searched using the PubMed database. Expert commentary: Development, optimization and validation of robust, affordable and simple high-throughput glycosylation analysis methods is essential for discovery and validation of diagnostic and prognostic glycan biomarkers. Although significant advances in glycosylation analysis have been made in recent years, currently used protocols will have to be further optimized to enable subsequent analysis of glycosylation on all levels with the limited initial sample and in the minimal amount of time, which is still a challenging task.

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., (© 2015 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Background: Glycobiology is an underexplored research area in inflammatory bowel disease (IBD), and glycans are relevant to many etiological mechanisms described in IBD. Alterations in N-glycans attached to the immunoglobulin G (IgG) Fc fragment can affect molecular structure and immunological function. Recent genome-wide association studies reveal pleiotropy between IBD and IgG glycosylation. This study aims to explore IgG glycan changes in ulcerative colitis (UC) and Crohn's disease (CD)., Methods: IgG glycome composition in patients with UC (n = 507), CD (n = 287), and controls (n = 320) was analyzed by ultra performance liquid chromatography., Results: Statistically significant differences in IgG glycome composition between patients with UC or CD, compared with controls, were observed. Both UC and CD were associated with significantly decreased IgG galactosylation (digalactosylation, UC: odds ratio [OR] = 0.71; 95% confidence interval [CI], 0.5-0.9; P = 0.01; CD: OR = 0.41; CI, 0.3-0.6; P = 1.4 × 10) and significant decrease in the proportion of sialylated structures in CD (OR = 0.46, CI, 0.3-0.6, P = 8.4 × 10). Logistic regression models incorporating measured IgG glycan traits were able to distinguish UC and CD from controls (UC: P = 2.13 × 10 and CD: P = 2.20 × 10), with receiver-operator characteristic curves demonstrating better performance of the CD model (area under curve [AUC] = 0.77) over the UC model (AUC = 0.72) (P = 0.026). The ratio of the presence to absence of bisecting GlcNAc in monogalactosylated structures was increased in patients with UC undergoing colectomy compared with no colectomy (FDR-adjusted, P = 0.05)., Conclusions: The observed differences indicate significantly increased inflammatory potential of IgG in IBD. Changes in IgG glycosylation may contribute to IBD pathogenesis and could alter monoclonal antibody therapeutic efficacy. IgG glycan profiles have translational potential as IBD biomarkers.