Immunoglobulin G N-glycome Research Articles

Glycan clock of ageing-analytical precision and time-dependent inter- and i-individual variability.

Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age.For precision analysis, three different plasma pools were analysed over 26days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10years, respectively. Samples were collected every 3months and 3weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits.The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits.The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths.

IgG glycomic profiling identifies potential biomarkers for diagnosis of echinococcosis

Echinococcosis caused by larval stage of the genus Echinococcus, is a serious and potentially fatal parasitic zoonosis distributed globally. The two types of the disease in human are cystic echinococcosis (CE) and alveolar echinococcosis (AE). As the biological and encysting characteristics of the parasite, early diagnosis remains to address. In the present study, we demonstrate the value of Immunoglobulin G (IgG) glycome as a potential diagnostic biomarker for echinococcosis. Serum IgG glycome profiles were analyzed by ultra-performance liquid chromatography in a cohort comprised of 127 echinococcosis patients, of them 98 were diagnosed as CE and 29 as AE. IgG N-glycome analysis in pretreatment serum of echinococcosis patients presents 25 glycans and 64 derived traits. Compared with IgG glycans of healthy control group, neutral glycans, fucosylation and agalactosylated N-glycans increased while sialylation and galactosylation decreased in echinococcosis patients. Combined with a machine-learning-based approach, we built three biomarker combinations to distinguish CE, AE and healthy controls. Meanwhile, galactosylation, sialylation and A2BG2S1 in IgG glycan profiles were evidently associated with different types of CE (from CE1 to CE5). Our findings suggest that the alterations in IgG N-glycome may be of value in CE and AE diagnosis and follow-up CE disease progress. The role of IgG N-glycans as diagnostic biomarker remains to be verified in future study.

Anti-TNF Biologicals Enhance the Anti-Inflammatory Properties of IgG N-Glycome in Crohn's Disease.

Crohn's disease (CD) is a chronic inflammation of the digestive tract that significantly impairs patients' quality of life and well-being. Anti-TNF biologicals revolutionised the treatment of CD, yet many patients do not adequately respond to such therapy. Previous studies have demonstrated a pro-inflammatory pattern in the composition of CD patients' immunoglobulin G (IgG) N-glycome compared to healthy individuals. Here, we utilised the high-throughput UHPLC method for N-glycan analysis to explore the longitudinal effect of the anti-TNF drugs infliximab and adalimumab on N-glycome composition of total serum IgG in 198 patients, as well as the predictive potential of IgG N-glycans at baseline to detect primary non-responders to anti-TNF therapy in 1315 patients. We discovered a significant decrease in IgG agalactosylation and an increase in monogalactosylation, digalactosylation and sialylation during the 14 weeks of anti-TNF treatment, regardless of therapy response, all of which suggested a diminished inflammatory environment in CD patients treated with anti-TNF therapy. Furthermore, we observed that IgG N-glycome might contain certain information regarding the anti-TNF therapy outcome before initiating the treatment. However, it is impossible to predict future primary non-responders to anti-TNF therapy based solely on IgG N-glycome composition at baseline.

Physical Exercise Induces Significant Changes in Immunoglobulin G N-Glycan Composition in a Previously Inactive, Overweight Population.

Regular exercise improves health, modulating the immune system and impacting inflammatory status. Immunoglobulin G (IgG) N-glycosylation reflects changes in inflammatory status; thus, we investigated the impact of regular exercise on overall inflammatory status by monitoring IgG N-glycosylation in a previously inactive, middle-aged, overweight and obese population (50.30 ± 9.23 years, BMI 30.57 ± 4.81). Study participants (N = 397) underwent one of three different exercise programs lasting three months with blood samples collected at baseline and at the end of intervention. After chromatographically profiling IgG N-glycans, linear mixed models with age and sex adjustment were used to investigate exercise effects on IgG glycosylation. Exercise intervention induced significant changes in IgG N-glycome composition. We observed an increase in agalactosylated, monogalctosylated, asialylated and core-fucosylated N-glycans (padj = 1.00 × 10-4, 2.41 × 10-25, 1.51 × 10-21 and 3.38 × 10-30, respectively) and a decrease in digalactosylated, mono- and di-sialylated N-glycans (padj = 4.93 × 10-12, 7.61 × 10-9 and 1.09 × 10-28, respectively). We also observed a significant increase in GP9 (glycan structure FA2[3]G1, β = 0.126, padj = 2.05 × 10-16), previously reported to have a protective cardiovascular role in women, highlighting the importance of regular exercise for cardiovascular health. Other alterations in IgG N-glycosylation reflect an increased pro-inflammatory IgG potential, expected in a previously inactive and overweight population, where metabolic remodeling is in the early stages due to exercise introduction.

Comparative analysis of transferrin and IgG N-glycosylation in two human populations

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans’ role in their distinct physiological functions.

IgG N-Glycosylation Is Altered in Coronary Artery Disease.

Coronary artery disease (CAD) is the most common cardiovascular disease (CVD), and previous studies have shown a significant association between N-glycosylation, a highly regulated posttranslational modification, and the development of atherosclerotic plaques. Our aim was to determine whether the N-glycome of immunoglobulin G (IgG) is associated with CAD, as N-glycans are known to alter the effector functions of IgG, which may enhance the inflammatory response in CAD. Therefore, in this study, we isolated IgG from subjects with coronary atherosclerosis (CAD+) and from subjects with clean coronaries (CAD-). The purified IgGs were denatured and enzymatically deglycosylated, and the released and fluorescently labelled N-glycans were analysed by ultra-high performance liquid chromatography based on hydrophilic interactions with fluorescence detection (HILIC-UHPLC-FLR). Sex-stratified analysis of 316 CAD- and 156 CAD+ cases revealed differences in IgG N-glycome composition. The most notable differences were observed in women, where the presence of sialylated N-glycan structures was negatively associated with CAD. The obtained chromatograms provide insight into the IgG N-glycome composition in CAD as well as the biomarker potential of IgG N-glycans in CAD.

The IgG glycome of SARS-CoV-2 infected individuals reflects disease course and severity.

Immunoglobulin G (IgG) antibodies play an important role in the immune response against viruses such as SARS-CoV-2. As the effector functions of IgG are modulated by N-glycosylation of the Fc region, the structure and possible function of the IgG N-glycome has been under investigation in relation to divergent COVID-19 disease courses. Through LC-MS analysis we studied both total IgG1 and spike protein-specific IgG1 Fc glycosylation of 129 German and 163 Brazilian COVID-19 patients representing diverse patient populations. We found that hospitalized COVID-19 patients displayed decreased levels of total IgG1 bisection and galactosylation and lowered anti-S IgG1 fucosylation and bisection as compared to mild outpatients. Anti-S IgG1 glycosylation was dynamic over the disease course and both anti-S and total IgG1 glycosylation were correlated to inflammatory markers. Further research is needed to dissect the possible role of altered IgG glycosylation profiles in (dys)regulating the immune response in COVID-19.

Effects of low-calorie and different weight-maintenance diets on IgG glycome composition.

Obesity-induced inflammation activates the adaptive immune system by altering immunoglobulin G (IgG) glycosylation in a way to produce more proinflammatory antibodies. The IgG glycome has already been well studied, and its alterations are correlated with a high body mass index (BMI) and central adiposity. Still, the IgG N-glycome susceptibility to different dietary regimes for weight control after the initial weight loss has not been studied. To explore changes in IgG glycosylation induced by weight loss and subsequent weight-maintenance diets, we analyzed 1,850 IgG glycomes from subjects in a dietary intervention Diogenes study. In this study, participants followed a low-calorie diet (LCD) providing 800 kcal/d for 8 weeks, followed by one of five weight-maintenance diets over a 6-month period. The most significant alteration of the IgG N-glycome was present 8 weeks after the subjects underwent an LCD, a statistically significant decrease of agalactosylated and the increase of sialylated N glycans. In the follow-up period, the increase in glycans with bisecting GlcNAc and the decrease in sialylated glycans were observed. Those changes were present regardless of the diet type, and we did not observe significant changes between different diets. However, it should be noted that in all five diet groups, there were individuals who prominently altered their IgG glycome composition in either proinflammatory or anti-inflammatory directions.

IgG N-Glycosylation from Patients with Pemphigus Treated with Rituximab.

Pemphigus is a life-threatening auto-immune blistering disease of the skin and mucous membrane that is caused by the production of auto-antibodies (auto-Abs) directed against adhesion proteins: desmoglein 1 and 3. We demonstrated in the “Ritux3” trial, the high efficacy of rituximab, an anti-CD20 recombinant monoclonal antibody, as the first-line treatment for pemphigus. However, 25% of patients relapsed during the six-month period after rituximab treatment. These early relapses were associated with a lower decrease in anti-desmoglein auto-Abs after the initial cycle of rituximab. The N-glycosylation of immunoglobulin-G (IgG) can affect their affinity for Fc receptors and their serum half-life. We hypothesized that the extended half-life of Abs could be related to modifications of IgG N-glycans. The IgG N-glycome from pemphigus patients and its evolution under rituximab treatment were analyzed. Pemphigus patients presented a different IgG N-glycome than healthy donors, with less galactosylated, sialylated N-glycans, as well as a lower level of N-glycans bearing an additional N-acetylglucosamine. IgG N-glycome from patients who achieved clinical remission was not different to the one observed at baseline. Moreover, our study did not identify the N-glycans profile as discriminating between relapsing and non-relapsing patients. We report that pemphigus patients present a specific IgG N-glycome. The changes observed in these patients could be a biomarker of autoimmunity susceptibility rather than a sign of inflammation.

IgG N-glycome changes during the course of severe COVID-19: An observational study

SummaryBackgroundThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a respiratory illness named coronavirus disease 2019 (COVID-19), which is one of the main global health problems since 2019. Glycans attached to the Fc portion of immunoglobulin G (IgG) are important modulators of IgG effector functions. Fc region binds to different receptors on the surface of various immune cells, dictating the type of immune response. Here, we performed a large longitudinal study to determine whether the severity and duration of COVID-19 are associated with altered IgG glycosylation.MethodsUsing ultra-high-performance liquid chromatography analysis of released glycans, we analysed the composition of the total IgG N-glycome longitudinally during COVID-19 from four independent cohorts. We analysed 77 severe COVID-19 cases from the HR1 cohort (74% males, median age 72, age IQR 25-80); 31 severe cases in the HR2 cohort (77% males, median age 64, age IQR 41-86), 18 mild COVID-19 cases from the UK cohort (17% males, median age 50, age IQR 26-71) and 28 mild cases from the BiH cohort (71% males, median age 60, age IQR 12-78).FindingsMultiple statistically significant changes in IgG glycome composition were observed during severe COVID-19. The most statistically significant changes included increased agalactosylation of IgG (meta-analysis 95% CI [0.03, 0.07], adjusted meta-analysis P= <0.0001), which regulates proinflammatory actions of IgG via complement system activation and indirectly as a lack of sialylation and decreased presence of bisecting N-acetylglucosamine on IgG (meta-analysis 95% CI [-0.11, -0.08], adjusted meta-analysis P= <0.0001), which indirectly affects antibody-dependent cell-mediated cytotoxicity. On the contrary, no statistically significant changes in IgG glycome composition were observed in patients with mild COVID-19.InterpretationThe IgG glycome in severe COVID-19 patients is statistically significantly altered in a way that it indicates decreased immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the severity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in COVID-19.FundingThis work has been supported in part by Croatian Science Foundation under the project IP-CORONA-2020-04-2052 and Croatian National Centre of Competence in Molecular Diagnostics (The European Structural and Investment Funds grant #KK.01.2.2.03.0006), by the UKRI/MRC (Cov-0331 - MR/V027883/1) and by the National Institutes for Health Research Nottingham Biomedical Research Centre and by Ministry Of Science, Higher Education and Youth Of Canton Sarajevo, grant number 27-02-11-4375-10/21.

Investigation of the causal relationships between human IgG N-glycosylation and 12 common diseases associated with changes in the IgG N-glycome.

Changes in the N-glycosylation of immunoglobulin G (IgG) are often observed in pathological states, such as autoimmune, inflammatory, neurodegenerative, cardiovascular diseases and some types of cancer. However, in most cases, it is not clear if the disease onset causes these changes, or if the changes in IgG N-glycosylation are among the risk factors for the diseases. The aim of this study was to investigate the casual relationships between IgG N-glycosylation traits and 12 diseases, in which the alterations of IgG N-glycome were previously reported, using two sample Mendelian randomization (MR) approach. We have performed two sample MR using publicly available summary statistics of genome-wide association studies of IgG N-glycosylation and disease risks. Our results indicate positive causal effect of systemic lupus erythematosus (SLE) on the abundance of N-glycans with bisecting N-acetylglucosamine in the total IgG N-glycome. Therefore, we suggest regarding this IgG glycosylation trait as a biomarker of SLE. We also emphasize the need for more powerful GWAS studies of IgG N-glycosylation to further elucidate the causal effect of IgG N-glycome on the diseases.

P–591 Mass spectrometry-based glycomic profiling of the follicular fluid total immunoglobulin G and proteome N-glycomes reveals deregulated inflammatory processes associated with specific controlled ovarian stimulation protocols

Abstract Study question Is there a significant inflammatory-related difference between analyzed follicular fluid (FF) glycome profiles regarding the ovarian stimulation protocol used in patients? Summary answer Observed differences between analyzed glycome profiles from patients that underwent different controlled ovarian stimulation (COS) protocols point to deregulated inflammatory processes associated with specific COS. What is known already Successful physiological folliculogenesis and ovulation require an adequate inflammatory response. On the other hand, COS application in ART relies on induced hormonal activation of systemic inflammatory processes. Several studies have confirmed a rise in inflammatory cytokines, CRP, and other markers of inflammation in patients subjected to different COS protocols, pointing to an enhanced inflammatory response during ovulation stimulation. Glycoproteins and glycans have an indisputable role in immune response modulation: proper glycosylation of glycoproteins plays a pivotal role in the regulation of normal physiological processes, and aberrant glycosylation of glycoproteins has been associated with various pathological states, including inflammation. Study design, size, duration Study design: Cross sectional – FFs from patients that underwent ART in modified natural cycle (MNC group) versus FFs from patients that underwent ART under GnRH antagonist COS (COS group). Size: 20 FFs from 20 patients undergoing ART. Duration: One year. Sampling procedure: Each FF was aspirated from the dominant follicle. In the COS group, only the fluid from the first aspirated follicle of each patient was collected. Participants/materials, setting, methods Study included 20 FF samples from 20 patients divided into two groups according to the applied ovarian stimulation protocol: MNC group (n = 10) and COS group (n = 10). The immunoglobulin G (IgG) was isolated from FF samples by immunoaffinity chromatography. The N-linked glycans derived from IgG molecule and the remaining FF total proteomes were enzymatically cleaved and subjected to derivatization procedure. N-glycomes of FF-isolated IgG and total proteomes were analyzed separately by MALDI-TOF-MS. Main results and the role of chance FF IgG N-glycome profiling The MALDI-TOF-MS based comparative analysis of the individual glycan relative abundances, revealed several significantly deregulated glycoforms between analyzed groups whose levels were significantly elevated (p˂0.05) in the COS vs. MNC group. Furthermore, additional low abundant N-glycan species were also found to be deregulated between the analyzed groups: two monogalactolysed and monosialylated N-glycan compositions were only identified in the COS group. The comparative analysis of FF IgG N-glycome features revealed statistically relevant differences in the levels of two derived traits: galactosylation and bigalactosylation levels of the FF IgG N-glycome, both significantly downregulated (p˂0.05) in the MNC vs. COS profile. Comparative analysis of FF total proteome N-glycome The majority of identified glycan compositions were complex type N-glycans representing more than 98% of the total N-glycome profiles in both analyzed groups. The comparative analysis of individual glycan relative abundances revealed relevant differences in regulation of ten N-glycan species between the two analyzed profiles. In the MNC group, six N-glycan species showed significantly increased abundances (p˂0.05) compared with the COS group. Moreover, two compositions were exclusively identified in the MNC group, while two compositions were identified only in the COS group. Limitations, reasons for caution Since this preliminary study was conducted on relatively small sample size, all results should be verified on a larger sample set. Moreover, focused glycosylation analysis of a panel of individual FF acute phase blood serum derived proteins and immunoglobulins, might additionally clarify the inflammatory mechanisms underlying different ART stimulation protocols. Wider implications of the findings: While glycome profiling of human FFs was conducted for the first time, previous evidence supports the shown association of aberrant inflammation in diverse ART stimulation protocols and in development of various pathological states (i.e. OHSS). Obtained results are in line with previous similar studies performed in the human plasma. Trial registration number uniri-biomed–18–161 1310

Glycosylation of IgG Associates with Hypertension and Type 2 Diabetes Mellitus Comorbidity in the Chinese Muslim Ethnic Minorities and the Han Chinese.

Objectives: Hypertension and type 2 diabetes mellitus comorbidity (HDC) is common, which confers a higher risk of cardiovascular disease than the presence of either condition alone. Describing the underlying glycomic changes of immunoglobulin G (IgG) that predispose individuals to HDC may help develop novel protective immune-targeted and anti-inflammatory therapies. Therefore, we investigated glycosylation changes of IgG associated with HDC. Methods: The IgG N-glycan profiles of 883 plasma samples from the three northwestern Chinese Muslim ethnic minorities and the Han Chinese were analyzed by ultra-performance liquid chromatography instrument. Results: We found that 12 and six IgG N-glycan traits showed significant associations with HDC in the Chinese Muslim ethnic minorities and the Han Chinese, respectively, after adjustment for potential confounders and false discovery rate. Adding the IgG N-glycan traits to the baseline models, the area under the receiver operating characteristic curves (AUCs) of the combined models differentiating HDC from hypertension (HTN), type 2 diabetes mellitus (T2DM), and healthy individuals were 0.717, 0.747, and 0.786 in the pooled samples of Chinese Muslim ethnic minorities, and 0.828, 0.689, and 0.901 in the Han Chinese, respectively, showing improved discriminating performance than both the baseline models and the glycan-based models. Conclusion: Altered IgG N-glycan profiles were shown to associate with HDC, suggesting the involvement of inflammatory processes of IgG glycosylation. The alterations of IgG N-glycome, illustrated here for the first time in HDC, demonstrate a biomarker potential, which may shed light on future studies investigating their potential for monitoring or preventing the progression from HTN or T2DM towards HDC.

The effect of n-3 polyunsaturated fatty acids-enriched hen eggs consumption on IgG and total plasma protein N-glycosylation in healthy individuals and cardiovascular patients.

This study determined the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs)-enriched hen eggs consumption on immunoglobulin G (IgG) and total plasma protein N-glycan profiles and inflammatory biomarkers level in healthy individuals (N=33) and cardiovascular (CV) patients (N=21). Subjects were divided to Control-Healthy and Control-CV subgroups [consumed three regular hens' eggs/daily (249 mg n-3 PUFAs/day)], and n-3 PUFAs-Healthy and n-3 PUFAs-CV subgroups [consumed three n-3 PUFAs-enriched hen eggs/daily (1053 mg n-3 PUFAs/day)] for 3weeks. Serum-free fatty acids profile and high-sensitivity C-reactive protein, interleukin 6 and 10 (IL-6, IL-10) and tumor necrosis factor alpha were measured. Total plasma protein and IgG N-glycome have been profiled before and after dietary protocols. Serum n-3 PUFAs concentration significantly increased following n-3 PUFAs hen eggs consumption in both n-3 PUFAs-Healthy and n-3 PUFAs-CV. IL-10 significantly increased in both Healthy subgroups, whereas no change occurred in CV subgroups. Derived IgG N-glycan traits: bisecting N-acetylglucosamine (B) significantly decreased in n-3 PUFAs-Healthy, whereas agalactosylation (G0) and core fucosylation (CF) significantly increased in Control-Healthy. Derived total plasma protein N-glycan traits: high branching glycans, trigalactosylation, tetragalactosylation, trisialylation, tetrasialylation and antennary fucosylation significantly decreased, whereas G0, monogalactosylation (G1), neutral glycans (S0), B, CF and oligomannose structures significantly increased in n-3 PUFAs-CV. Digalactosylation significantly decreased, and G0, G1, S0, disialylation, B and CF significantly increased in Control-CV. n-3 PUFAs consumption alters IgG N-glycan traits and IL-10 in healthy individuals, and total plasma protein N-glycan traits in CV patients, by shifting them toward less inflammatory N-glycosylation profile.

Robustness and repeatability of GlycoWorks RapiFluor-MS IgG N-glycan profiling in a long-term high-throughput glycomic study.

Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here, we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a 5-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three-fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2%. The highest CVs ranging from 16 to 29% accompanied low abundance glycan peaks with the individual relative peak area below 1% that together made up for <2% of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

Glycosylation and Aging.

Human lifespan has increased significantly in the last 200years, emphasizing our need to age healthily. Insights into molecular mechanisms of aging might allow us to slow down its rate or even revert it. Similar to aging, glycosylation is regulated by an intricate interplay of genetic and environmental factors. The dynamics of glycopattern variation during aging has been mostly explored for plasma/serum and immunoglobulin G (IgG) N-glycome, as we describe thoroughly in this chapter. In addition, we discuss the potential functional role of agalactosylated IgG glycans in aging, through modulation of inflammation level, as proposed by the concept of inflammaging. We also comment on the potential to use the plasma/serum and IgG N-glycome as a biomarker of healthy aging and on the interventions that modulate the IgG glycopattern. Finally, we discuss the current knowledge about animal models for human plasma/serum and IgG glycosylation and mention other, less explored, instances of glycopattern changes during organismal aging and cellular senescence.

IgG N-glycans.

Glycosylation, one of the most common post-translational modifications in mammalian cells, impacts many biological processes such as cell adhesion, proliferation and differentiation. As the most abundant glycoprotein in human serum, immunoglobulin G (IgG) plays a vital role in immune response and protection. There is a growing body of evidence suggests that IgG structure and function are modulated by attached glycans, especially N-glycans, and aberrant glycosylation is associated with disease states. In this chapter, we review IgG glycan repertoire and function, strategies for profiling IgG N-glycome and recent studies. Mass spectrometry (MS) based techniques are the most powerful tools for profiling IgG glycome. IgG glycans can be divided into high-mannose, biantennary complex and hybrid types, modified with mannosylation, core-fucosylation, galactosylation, bisecting GlcNAcylation, or sialylation. Glycosylation of IgG affects antibody half-life and their affinity and avidity for antigens, regulates crystallizable fragment (Fc) structure and Fcγ receptor signaling, as well as antibody effector function. Because of their critical roles, IgG N-glycans appear to be promising biomarkers for various disease states. Specific IgG glycosylation can convert a pro-inflammatory response to an anti-inflammatory activity. Accordingly, IgG glycoengineering provides a powerful approach to potentially develop effective drugs and treat disease. Based on the understanding of the functional role of IgG glycans, the development of vaccines with enhanced capacity and long-term protection are possible in the near future.

Profiling of IgG N-glycome during mouse aging: Fucosylated diantennary glycans containing one Neu5Gc-linked LacNAc are associated with age

N-glycosylation of immunoglobulin G (IgG) has been reported to change in human aging and in some age-related diseases. To further understand the molecular processes that determine these alterations, a detailed examination of individual IgG N-glycans with aging remains required. Mouse is the most commonly used model animal in studies of aging and age-related diseases, and mice have the advantage of relatively controllable genetic and environment variations compared to human. In this study, we systemically investigated the changes in serum IgG N-glycome in C57BL/6 mice during aging at 12 time points (6–80 weeks) via ultraperformance liquid chromatography with fluorescence detection. The study demonstrated several important findings. First, four chromatographic IgG N-glycan peaks were identified for the first time, including a high-mannose glycan, a monoantennary glycan, and two afucosylated glycans. Second, most of the IgG glycan levels changed significantly and presented pronounced gender-related differences from 6 to 12 weeks. Interestingly, all the IgG glycan levels tended to be similar between male and female mice at 12 weeks. Third, the level of fucosylated diantennary glycans containing one N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) decreased gradually and showed a significant negative correlation with age from 24 to 80 weeks (r = −0.716, p < 0.0001), which was not sex-specific. SignificanceMore comprehensive profile of murine IgG N-glycans by ultraperformance liquid chromatography with fluorescence detection was shown in this study with four newly identified chromatographic murine IgG N-glycan peaks. The majority of IgG N-glycans showed substantial stage-specific changes and sex-related differences during mouse aging, indicating a strict regulatory mechanism of glycan synthesis. The level of fucosylated diantennary glycans containing one Neu5Gc-linked LacNAc was significantly negatively correlated with age from 24 to 80 weeks, suggesting its great potential as an aging biomarker. The detailed characteristics of IgG N-glycosylation with aging in C57BL/6 mice demonstrated in the present study could provide essential reference data for studying the function and mechanism of IgG glycosylation in age-related researches based on C57BL/6 mouse models.

Serum immunoglobulin G N-glycome: a potential biomarker in endometrial cancer.

BackgroundWith the increase in incidence and mortality of endometrial cancer (EC), there is an urgent need to explore non-invasive strategies for identifying EC patients and facilitating risk stratification. The alteration of immunoglobulin G (IgG) N-glycome has been indicated in autoimmune diseases and several cancer types, demonstrating a significant association with disease pathogenesis and progression. However, little has been investigated in the IgG N-glycome of EC patients.MethodsA total of 94 EC patients and 112 healthy females were recruited and sorted into an EC cohort and a control cohort. Serum samples were obtained from every participant, and IgG N-glycome profiling was conducted using ultra-performance liquid chromatography (UPLC).ResultsA total of 24 directly measured N-glycans and 11 derived traits based on the shared glycan structures were analyzed in the EC and control cohorts. We detected a significant downregulation of galactosylation and sialylation in the EC cohort compared with the control cohort, while glycans with bisecting N-acetylglucosamine (GlcNAc) were elevated in EC patients. Receiver operating characteristic (ROC) analysis based on glycan traits showed good diagnostic performance of IgG N-glycans for EC. Furthermore, by exploring the association of IgG N-glycome with prognostic risk factors in EC, we observed that lower levels of galactosylation and sialylation were correlated with high-risk factors including older age, non-endometrioid histologic subtypes, advanced stage, poor differentiation of tumor, and >50% myometrial invasion (MI).ConclusionsOur results suggest that the IgG N-glycome profile could be a potential biomarker for EC diagnosis and a promising indicator for prognostic risk factors, and thus may facilitate the early detection of EC and the identification of high-risk patients.

Association of the IgG N-glycome with the course of kidney function in type 2 diabetes

IntroductionInflammatory processes are thought to be involved in kidney function decline in individuals with type 2 diabetes. Glycosylation of immunoglobulin G (IgG) is an important post-translation process affecting the inflammatory...