Immunoglobulin G Glycosylation Research Articles

Local Immune Activation and Age Impact on Humoral Immunity in Mice, with a Focus on IgG Sialylation.

Age alters the host's susceptibility to immune induction. Humoral immunity with circulating antibodies, particularly immunoglobulin G (IgG), plays an essential role in immune response. IgG glycosylation in the fragment crystallizable (Fc) region, including sialylation, is important in regulating the effector function by interacting with Fc gamma receptors (FcγRs). Glycosylation is fundamentally changed with age and inflammatory responses. We aimed to explore the regulation of humoral immunity by comparing responses to antigen-induced immune challenges in young and adult mice using a local antigen-induced arthritis mouse model. This study examines the differences in immune response between healthy and immune-challenged states across these groups. Our initial assessment of the arthritis model indicated that adult mice presented more severe knee swelling than their younger counterparts. In contrast, we found that neither histological assessment, bone mineral density, nor the number of osteoclasts differs. Our data revealed an age-associated but not immune challenge increase in total IgG; the only subtype affected by immune challenge was IgG1 and partially IgG3. Interestingly, the sialylation of IgG2b and IgG3 is affected by age and immune challenges but not stimulated further by immune challenges in adult mice. This suggests a shift in IgG towards a pro-inflammatory and potentially pathogenic state with age and inflammation.

Are Changes in Serum IgG Glycosylation Related to the Severe Course of SARS-CoV-2 Infection and Recovery Process? In Search of New Diagnostic and Prognostic Biomarkers.

Immunoglobulin G (IgG) glycosylation affects its effector functions and is essential in many steps of the inflammatory cascade. Therefore, it may be an important parameter for assessing the body's immune response during the course of COVID-19 (Coronavirus disease 2019). The N- and O-glycosylation of serum IgG in severe COVID-19 patients (n=87), convalescents (n=50), and healthy subjects (n=65) were examined using a modified lectin-ELISA method with specific biotinylated lectins. The obtained data were analyzed using STATISTICA 13.3PL software. We showed significantly higher expression of Lewisx oligosaccharide structures in severe COVID-19 patients than in the other two groups. Moreover, significantly lower expression of Lewisy sugar structures in IgG glycans was observed in the convalescents when compared with COVID-19 patients and healthy subjects. The lowest expression of highly branched N-glycans in cases of severe COVID-19 indicates that the development of the disease is associated with the presence of typical IgG biantennary N-glycans. The lack of significant differences in the expression of Tn antigen in IgG between studied groups and the significantly lower expression of T antigen in convalescents compared to the patients with severe COVID-19 and healthy subjects indicates a decrease in the content of the T antigen in IgG O-glycans in subjects recovered from COVID-19. Substantially higher reactivities of IgG O-glycans with Jacalin observed in COVID-19 patients and convalescents in comparison to the control group were most probably caused by increased expression of core 3 O-glycans in IgG. Severe COVID-19 is accompanied by the expression in serum IgG of sialylated biantennary and highly branched N-glycans, decorated by fucose of Lewisx and Lewisy structures. The higher reactivity of IgG O-glycans with Jacalin in severe COVID-19 patients and convalescents indicates that the disease development and the recovery process are most probably accompanied by increased expression of the core 3 O-glycans.

GlYcoLISA: antigen-specific and subclass-specific IgG Fc glycosylation analysis based on an immunosorbent assay with an LC-MS readout.

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation modulates effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Consequently, assessing IgG Fc glycosylation is important for understanding the role of antibodies in infectious, alloimmune and autoimmune diseases. GlYcoLISA determines the Fc glycosylation of antigen-specific IgG by an immunosorbent assay with a liquid chromatography-mass spectrometry (LC-MS) readout. Detection of antigen-specific IgG glycosylation in a subclass- and site-specific manner is realized by LC-MS-based glycopeptide analysis after proteolytic cleavage. GlYcoLISA addresses challenges related to the low abundance of specific IgG and the high background of total IgG by using well-established immunosorbent assays for purifying antibodies of the desired specificity using immobilized antigen. Alternative methods with sufficient glycan resolution lack these important specificities. GlYcoLISA is performed in a 96-well plate format, and the analysis of 160 samples takes ~5 d, with 1 d for sample preparation, 2 d of LC-MS measurement and 2 d for partially automated data processing. GlYcoLISA requires expertise in LC-MS operation and data processing.

IgG sialylation occurs in B cells pre antibody secretion.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

An integrated method for IgG N-glycans enrichment and analysis: Understanding the role of IgG glycosylation in diabetic foot ulcer

Diabetic foot ulcer (DFU) is the most common and serious complication of diabetes, and its incidence, disability, and mortality rates are increasing worldwide. The pathogenesis of DFU is associated with dysregulated inflammation mediated by abnormal immunoglobulin G (IgG) glycosylation. In this study, we developed a comprehensive method for IgG N-linked glycosylation in the serum of DFU patients. Through analysis, we identified 31 IgG1 glycans, 32 IgG2 glycans, and 30 IgG4 glycans in the DFU serum. Furthermore, 13 IgG1 glycans, 12 IgG2 glycans, and 5 IgG4 glycans in the DFU groups were found to be significantly different from those of the control groups (p < 0.05). Of these, compared with the control group, one glycan was unique to DFU patients, and seven glycans were not detected in the DFU group. In terms of glycan characteristics, we observed a substantial decrease in galactosylation, sialylation and bisecting GlcNAcylation, and a significant increase in agalactosylation. Abnormal IgG N-glycosylation modifications were significantly associated with the chronic inflammation that is characteristic of DFU. Further, this is the first comprehensive analysis of subclass-specific IgG N-glycosylation in DFU patients, which not only fills the gap of DFU in terms of the pathological mechanisms related to IgG glycosylation but also may provide valuable clues for the immunotherapeutic pathway of DFU.

Mapping of the gene network that regulates glycan clock of ageing.

Glycans are an essential structural component of immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation), a trait which was shown to be associated with ageing. We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in the in vitro IgG expression system. Upregulation of three genes, EEF1A1, MANBA and TNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in this in vitro expression system.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Immunomodulation of Antibody Glycosylation through the Placental Transfer

Establishing an immune balance between the mother and fetus during gestation is crucial, with the placenta acting as the epicenter of immune tolerance. The placental transfer of antibodies, mainly immunoglobulin G (IgG), is critical in protecting the developing fetus from infections. This review looks at how immunomodulation of antibody glycosylation occurs during placental transfer and how it affects fetal health. The passage of maternal IgG antibodies through the placental layers, including the syncytiotrophoblast, stroma, and fetal endothelium, is discussed. The effect of IgG subclass, glycosylation, concentration, maternal infections, and antigen specificity on antibody transfer efficiency is investigated. FcRn-mediated IgG transport, influenced by pH-dependent binding, is essential for placental transfer. Additionally, this review delves into the impact of glycosylation patterns on antibody functionality, considering both protective and pathological effects. Factors affecting the transfer of protective antibodies, such as maternal vaccination, are discussed along with reducing harmful antibodies. This in-depth examination of placental antibody transfer and glycosylation provides insights into improving neonatal immunity and mitigating the effects of maternal autoimmune and alloimmune conditions.

Specific IgG glycosylation differences precede relapse in PR3-ANCA associated vasculitis patients with and without ANCA rise.

Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares. Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV. Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis (P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P = 0.02). Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre.

Use of single analytic tool to quantify both absolute N-glycosylation and glycan distribution in monoclonal antibodies.

Recombinant proteins represent almost half of the top selling therapeutics-with over a hundred billion dollars in global sales-and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N-glycan micro- and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP-GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.

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.

Mouse milk immunoglobulin G Fc-linked N-glycosylation nano-LC–MS analysis in a model of vancomycin exposure during pregnancy

Breast milk immunoglobulin G (IgG) plays an important role in the transfer of passive immunity in early life and in shaping the neonatal immune system through N-glycan-mediated effector functions. Currently, there are no protocols available to analyze breast milk IgG-Fc glycosylation in mouse models. Therefore, we developed and validated a glycoproteomic workflow for the medium-throughput subclass-specific nano-LC-MS analysis of IgG enriched from small milk volumes of lactating mice. With the established methods, the IgG glycopatterns in a mouse model of antibiotic use during pregnancy and increased asthma susceptibility in the offspring were analyzed. Pregnant BALB/c mice were treated with vancomycin during gestation days 8-17 and IgG1F, IgG2, and IgG3-Fc glycosylation was subsequently analyzed in maternal serum, maternal breast milk, and offspring serum on postnatal day 15. The IgG glycosylation profiles of mouse maternal milk and serum revealed no significant differences within the glycoforms quantified across subclasses. However, vancomycin use during pregnancy was associated with changes in IgG-Fc glycosylationin offspring serum, shown by the decreased relative abundance of the IgG1F-G1 and IgG3-G0 glycoforms, together with the increased relative abundance of the IgG3-G2 and S1 glycoforms. The workflow presented will aid in the emerging integrative multi-omics- and glycomics-oriented milk analyses both in rodent models and human cohorts for a better understanding of mother-infant immunological interactions.

Do I Need to Trypsin Digest Before Releasing IgG Glycans With PNGase-F?

Immunoglobulin G (IgG) is the main immunoglobulin in human serum, and its biological activity is modulated by glycosylation on its fragment crystallizable region. Glycosylation of IgGs has shown to be related to aging, disease progression, protein stability, and many other vital processes. A common approach to analyze IgG glycosylation involves the release of the N-glycans by PNGase F, which cleaves the linkage between the asparagine residue and the innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the core GlcNAc. The biological significance of these glycans necessitates the development of accurate methods for their characterization and quantification. Currently, researchers either perform PNGase F deglycosylation on intact or trypsin-digested IgGs. Those who perform PNGase F deglycosylation on trypsin-digested IgGs argue that proteolysis is needed to reduce steric hindrance, whereas the other group states that this step is not needed, and the proteolytic step only adds time. There is minimal experimental evidence supporting either assumption. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this deglycosylation reaction for intact IgGs and IgG glycopeptides. Statistically significant differences in the rate of deglycosylation performed on intact IgGs and trypsin-digested IgGs were determined, and the rate of PNGase F deglycosylation on trypsin-digested IgGs was found to be 3- to 4-times faster than on intact IgG.

Abstract 40: Score Based on Immunoglobulin G N-glycans for Cardiovascular Risk Prediction: Secondary Analysis of Two Randomized Trials of Primary and Secondary Prevention

Introduction: Immunoglobulin G (IgG) are post-translationally modified proteins with the addition of complex carbohydrate molecules (glycans). These glycans can modulate IgG inflammatory capacity and determine the transition from healthy to diseased tissue. Hypothesis: A glycan score based on certain IgG glycosylation patterns is associated with CVD risk and can improve model prediction. Methods: IgG glycosylation profiles were measured on baseline plasma samples from nested CVD case-control participants from 2 studies: JUPITER (NCT00239681; Npairs=162; discovery) and the TNT (NCT00327691; Npairs=367, validation). Lasso regression was used to select IgG glycan peaks (GPs). The linear combination of selected IgG-GPs that significantly associated with CVD in a mutually adjusted conditional regression comprised the glycan score (IgG GS ). CVD prediction using IgG GS was investigated controlling for clinical risk factors. Using a parametric approach, we calculated the area under the curve (AUC) with and without IgG GS . Results: From 24 IgG-GPs, Lasso selected 4 IgG-GPs that were also associated with incident CVD (P&lt;.05) in a mutually adjusted conditional logistic regression. These 4 IgG-GPs (9, 12, 19, 20) composed the IgG glycan score (IgG GS ; Fig1A), which was significantly associated with incident CVD in JUPITER after adjusting for clinical risk factors (model 2 hazard ratio [HR]: 2.1, 95% CI 1.55 - 2.84), with significant validation in TNT (HR = 1.2, 95% CI 1.03 - 1.4; Fig. 1B). The AUC was higher for the model with IgG GS (0.74, 95%CI = 0.69 - 0.81) than without (0.69, 95%CI = 0.67 - 0.71) in JUPITER; replicating in TNT: 0.72 [0.71 - 0.83] vs 0.64, [0.62 - 0.65]. The P-value for the likelihood ratio test comparing models with and without IgG GS was 5.9x10 -8 in JUPITER and .02 in TNT. Conclusions: An IgG glycan score with 4 IgG N-glycans was positively associated with incident CVD in the JUPITER primary prevention population, which replicated in TNT, a secondary prevention cohort, and improved model prediction performance.

Abstract 11057: Glycosylation Profile of Immunoglobulin G and Incident Cardiovascular Events: Secondary Analysis of Two Randomized Clinical Trials

Introduction: Alterations in the glycosylation pattern of immunoglobulin G (IgG) play a key role in the transition from healthy to diseased tissue, and have been related to inflammation, immune function, and cardiovascular disease (CVD) risk factors. Hypothesis: We hypothesized that IgG glycosylation patterns are associated with the risk of developing CVD events. Methods: IgG glycosylation profiles were measured using ultra-performance liquid chromatography on baseline plasma samples from two nested CVD case-control studies within the JUPITER (NCT00239681; Npairs=162; discovery) and the TNT (Npairs=367, validation) randomized trials. In both cohorts, we used conditional logistic regression adjusted for age, sex, statin, race, LDLC, smoking, hypertension, BMI, HDLC, triglycerides, and hs -CRP to examine the association of 24 IgG glycan peaks (IgG-GPs) with incident CVD events. In the controls sample, we performed Spearman correlation between IgG-GPs and hs -CRP, a biomarker of inflammation. Results: After adjusting for CVD risk factors and correcting for multiple testing (overall FDR&lt;.05), one agalactosylated fucosylated glycan (IgG-GP4) was positively associated with CVD risk (odds ratio [OR]=1.4, 95%CI= 1.1; 1.9), while three digalactosylated (IgG-GPs 12, 13, and 14), and one monosialylated glycan (IgG-GP 16) were negatively associated with CVD risk in JUPITER (OR ranging from 0.7 to 0.8, 95%CI=0.5; 1.0). The results replicated in TNT (Figure 1). IGG-GP4 positively correlated with hs -CRP, while an inverse non-significant correlation, was observed for IGG-GPs 12, 13, and 14 with hsCRP (Figure 1). Conclusions: Certain glycosylation traits were associated with risk of incident CVD independent of clinical risk factors and statin therapy. Specifically, one agalactosylated fucosylated glycan was positively associated with CVD risk, while digalactosylated and sialylated glycans were inversely associated with CVD risk in two independent cohorts.

Impact of cancer stem cell markers Nestin and Sox2 on invasion abilities of epithelial and mesenchymal pancreatic cancer cells

Immunoglobulin G (IgG) antibodies function, in part, through ligation of cell-surface Fc receptors such as FcγRIIIA (also known as CD16A). IgG glycosylation is known to impact antibody function, but the role of FcγRIIIA glycans, if any, is unclear. Patel et al. now reveal that these glycans do impact protein conformation and IgG affinity and display cell-specific glycosylation patterns, leading to a potential model in which the affinity and possibly function of Fc receptors is dictated by the cell type and its surface glycome.

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.

IgG Glycosylation Profiling of Peripheral Artery Diseases with Lectin Microarray.

Background: Inflammation plays a key role in the progression of atherosclerotic plaque for peripheral artery disease (PAD). Immunoglobulin G (IgG) glycosylation could modulate immunological effector functions and has been explored as biomarkers for various diseases. Methods: Lectin microarray was applied to analyze the expression profile of serum IgG glycosylation in patients with lower-extremity peripheral artery disease (LEPAD), carotid artery stenosis (CAS), abdominal aortic aneurysm (AAA), and healthy controls. Lectin blot was performed to validate the differences. Results: SNA (Sambucus nigra agglutinin) binding (preferred sialic acid) was significantly higher in the LEPAD (3.21 ± 2.06) and AAA (3.34 ± 2.42) groups compared to the CAS (2.47 ± 1.45) group. Significantly higher binding levels of ConA (Concanavalin A) (preferred mannose) and PSA (Pisum sativum agglutinin) (preferred fucose) were also observed in LEPAD compared to CAS patients. Among LEPAD patients, a significant lower binding level of Black bean crude (preferred GalNAc) was present for dyslipidemia patients. A higher binding level of MNA-M (Morniga M agglutinin) (preferred Mannose) and Jacalin-AIA (Artocarpus integrifolia agglutinin) (preferred Galβ3GalNAc) was observed for Fontaine severe patients. Higher binding levels of PHA-E (Phaseolus vulgaris Erythroagglutinin) and PHA-L (Phaseolus vulgaris Leucoagglutinin) (preferred Galβ4GlcNAc) were observed for diabetic patients, and higher binding of ASA (Allium sativum agglutinin) (preferred Mannose) was present in patients with hypertension. The level of high-sensitivity C-reactive protein (hsCRP) was positively associated with LTL (Lotus tetragonolobus lectin) (r = 0.44), PSA (r = 0.44), LCA (Lens Culinaris agglutinin) (r = 0.39), SNA (r = 0.57), and CSA (Cytisus sscoparius agglutinin) (r = 0.56). For CAS, symptomatic patients had lower binding levels of AAL (Aleuria aurantia lectin) (preferred fucose) and IAA (Iberis amara agglutinin) (preferred GalNAc). Blood total cholesterol level was positively associated with SNA-I (r = 0.36) and SBA (Soybean agglutinin) (r = r = 0.35). Creatinine levels were positively associated with lectins including, but not limited to, MNA-M (r = 0.42), CSA (r = 0.45), GHA (Glechoma hederacea agglutinin) (r = 0.42), and MNA-G (Morniga G agglutinin) (r = 0.45). Conclusion: LEPAD patients had increased IgG binding levels of SNA and ConA compared to CAS, which could provide potential diagnostic value. Fontaine severity was associated with Mannose-rich IgG N-glycan, while diabetic LEPAD correlated with bisecting GlcNAc. The levels of hsCRP and creatinine were positively associated with IgG fucosylation and galactosylation. Changes in IgG glycosylation may play important roles in PAD pathogenesis and progression.

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.

Differences in Immunoglobulin G Glycosylation Between Influenza and COVID-19 Patients.

The essential role of immunoglobulin G (IgG) in immune system regulation and combatting infectious diseases cannot be fully recognized without an understanding of the changes in its N-glycans attached to the asparagine 297 of the Fc domain that occur under such circumstances. These glycans impact the antibody stability, half-life, secretion, immunogenicity, and effector functions. Therefore, in this study, we analyzed and compared the total IgG glycome—at the level of individual glycan structures and derived glycosylation traits (sialylation, galactosylation, fucosylation, and bisecting N-acetylglucosamine (GlcNAc))—of 64 patients with influenza, 77 patients with coronavirus disease 2019 (COVID-19), and 56 healthy controls. Our study revealed a significant decrease in IgG galactosylation, sialylation, and bisecting GlcNAc (where the latter shows the most significant decrease) in deceased COVID-19 patients, whereas IgG fucosylation was increased. On the other hand, IgG galactosylation remained stable in influenza patients and COVID-19 survivors. IgG glycosylation in influenza patients was more time-dependent: In the first seven days of the disease, sialylation increased and fucosylation and bisecting GlcNAc decreased; in the next 21 days, sialylation decreased and fucosylation increased (while bisecting GlcNAc remained stable). The similarity of IgG glycosylation changes in COVID-19 survivors and influenza patients may be the consequence of an adequate immune response to enveloped viruses, while the observed changes in deceased COVID-19 patients may indicate its deviation.