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Kao, Chih-Chin, San-Yuan Wang, Yung-Kun Chuang, Wei-Yuan Lee, Wei-Chiao Chang, Mai-Szu Wu, Tai-Chih Kuo, and I.-Lin Tsai. "Clinical Mass Spectrometry Discovered Human IgG Sialylation as a Potential Biosignature for Kidney Function." Journal of Personalized Medicine 11, no. 8 (July 31, 2021): 761. http://dx.doi.org/10.3390/jpm11080761.
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Immunoglobulin G (IgG) N-glycosylation was discovered to have an association with inflammation status, which has the potential to be a novel biomarker for kidney diseases. In this study, we applied an ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method to plasma and urine samples from 57 individuals with different levels of kidney function. Natural abundances of total IgG, IgG1, IgG2, and IgG3 subclasses in plasma showed positive correlations to the estimated glomerular filtration rates (eGFRs). Eighteen IgG glycopeptides also showed positive correlations. In contrast, higher IgG amounts were found in urine samples from participants with lower eGFR values. After normalizing IgG glycopeptides from plasma to their respective protein amounts, H4N4F1S1-IgG1 (r = 0.37, p = 0.0047, significant) and H5N4F1S1-IgG1 (r = 0.25, p = 0.063, marginally significant) were the two glycopeptides that still had positive correlations with eGFRs. The results showed that the UHPLC-MS/MS method is capable of investigating IgG profiles, and monitoring IgG and glycosylation patterns is worthy of further clinical application for kidney disease.
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Pfeifle, R., J. Kittler, M. Wuhrer, G. Schett, and G. Krönke. "AB0016 THE IMPACT OF IL-17A THERAPY ON IGG SIALYLATION IN HUMANS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 1042.1–1043. http://dx.doi.org/10.1136/annrheumdis-2021-eular.1087.
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Background:Rheumatoid arthritis (RA) is characterized by autoreactive B- and T cells. Autoantibodies are a hallmark of RA and contribute to synovial inflammation. We have recently demonstrated that Th17 cells suppress the enzyme ST6 a-galactoside b-2,6-sialyltransferase (ST6GAL1) in developing plasma cells. Thereby, Th17 cells regulate the degree of autoantibody sialylation leading to the increased inflammatory activity of autoantibodies. These events correlate with the onset of RA, arguing for a crucial role of the IL-23/Th17 axis during the transition of asymptomatic autoimmunity into active RA. Therefore, treatment against the IL-23/TH17-axis might present an attractive therapeutic approach to halt or delay RA’s onset. However, the effects of Th17 cytokines like IL-17 on IgG glycosylation in humans are so far poorly studied.Objectives:To explore whether anti-IL17A treatment can inhibit pro-inflammatory IgG glycosylation patterns in humans.Methods:Total IgG from patient cohorts suffering from psoriatic arthritis (PsA) treated with Secukinumab (anti-IL-17 blockade, n=26) or Ustekinumab (anti-IL12/23 blockade, n=14) was compared with patients treated with anti-TNFa blockade as a control (n=20). The cohorts were age- and sex-matched and included patients being on therapy for at least six months. Total IgG was isolated using Protein G columns, and IgG glycopeptides of IgG1, IgG2, and IgG4 were analyzed using the LC-MS technique. The effect of IL-17 depletion on IgG glycosylation was analyzed in psoriatic arthritis patients who have been treated with secukinumab for at least six months. Furthermore, in a longitudinal approach, IgG1, IgG2, and IgG4 glycosylation were analyzed from samples, isolated before the beginning of anti-IL-17 blockade and after at least six months of therapy (n=16).Results:Cross-sectional comparison of cohorts treated with Ustekinumab, Sekukinumab, and anti-TNFa therapy did not show any significant differences in sialylation, galactosylation, or fucosylation of IgG1 and IgG2. IgG4 from anti-TNFa treated patients displayed a small increase of sialylation when compared to the Ustekinumab treated cohort.Longitudinal analyses, however, showed that IL-17A blockade during Secukinumab therapy caused a significant increase of sialic acid-rich IgG glycoforms on IgG1, IgG2 IgG4 patients, while the galactosylation, fucosylation remained unaffected.Conclusion:This data indicates that IL-17A blockade specifically affects IgG sialylation, while other Fc-glycan modifications remain unaltered. This data confirms our recent findings in mice, where cytokines of the IL-23/Th17 axis specifically induce the production of hypo-sialylated, proinflammatory autoantibodies in rheumatoid arthritis (RA) [2]. Therefore, neutralizing IL-17 might be a therapeutic option during the asymptomatic autoimmune prodromal phase in autoimmune diseases like RA, where TH17 cytokines orchestrate the emergence of a pro-inflammatory autoantibody response and the transition into active RA.References:[1]McInnes IB, G. Schett, The pathogenesis of rheumatoid arthritis. N Engl J Med 2011; 365: 2205-19.[2]Pfeifle R et al, Regulation of autoantibody activity by the IL-23-Th17 axis determines the onset of autoimmune disease. Nat Immunol. 2017, Jan;18(1):104-113.Disclosure of Interests:Rene Pfeifle Grant/research support from: Novartis AG., Julia Kittler: None declared, Manfred Wuhrer: None declared, Georg Schett: None declared, Gerhard Krönke Grant/research support from: Novartis AG
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Kozłowska, Kamila, Magdalena Rydlewska, Marta Ząbczyńska, and Ewa Pocheć. "IgG glycosylation in autoimmune diseases." Postępy Higieny i Medycyny Doświadczalnej 72 (November 12, 2018): 975–90. http://dx.doi.org/10.5604/01.3001.0012.7351.
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Immunoglobulin G (IgG) is the most abundant glycoprotein in human serum. All IgG subclasses have a single-conserved N-linked glycosylation site at Asn297 of the heavy chain and 10–30% of IgGs are N-glycosylated also in a Fab region. N-glycans of Fc are sialylated and fucosylated biantennary complex-type structures. Glycosylation plays a key role in antibody function, and IgG N-glycans are essential for the proper activity of the immune system. Fc glycans are important for IgG effector functions, whereas Fab oligosaccharides modulate antigen binding. Glycosylation changes of IgG are associated with the development of various human diseases, including autoimmune states. The modification of one sugar moiety in N-glycan structure may result in the stimulation or suppression of immune response. The lack of core fucose leads to the enhancement of pro-inflammatory activity, whereas an increase of sialylation determines immunosuppressive properties of IgG. The contribution of IgG Fc glycosylation changes has been demonstrated in the pathogenesis of rheumatoid arthritis, lupus erythematosus and Crohn’s disease. A decrease in IgG galactosylation and sialylation, found in these diseases, activates effector cells and triggers inflammatory reactions. A detailed analysis of changes in IgG glycosylation and their effects on the development of autoimmune diseases is important in the treatment of these diseases. IgGs with modified α2,6-sialylation are used as therapeutic antibodies with anti-inflammatory properties. Numerous studies on IgG glycosylation have provided evidence of the role of this post-translational modification in the proper functioning of antibodies and the importance of changes in the structure of IgG glycans, mainly incomplete galactosylation and desialylation, in the pathogenesis of many diseases. The continuation of these studies may contribute to explaining the mechanisms of autoimmunity that is still poorly understood.
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Hahm, Lee, and Ahn. "Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein." Molecules 24, no. 21 (October 30, 2019): 3924. http://dx.doi.org/10.3390/molecules24213924.
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A biosimilar fusion protein VEGFR-IgG consisting of vascular endothelial growth factor receptors 1 and 2 (VEGFR-1, VEGFR-2) and the Fc portion of human IgG1 was prepared for this study. The prepared fusion protein was expected to possess a total of five N-linked glycosylation sites: two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region. For site-specific glycan analysis, the fusion protein was hydrolyzed with trypsin, and the resulting tryptic digests were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The expected N-linked glycosylation sites were successfully identified and site-specific glycopeptide mapping was completed by Integrated GlycoProteome Analyzer (I-GPA) for the resulting raw tandem mass data. Finally, it was clearly confirmed that N-linked glycans for each glycosylation site showed significantly different patterns in microheterogeneity, which may indicate certain functions for each glycosylation site in the protein. Based on the mapping results, the unique features in glycan microheterogeneity for the five glycosylation sites of VEGFR-IgG fusion protein were compared site-specifically and further discussed to understand the functional meaning of each glycosylation pattern.
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Shadrina, Alexandra S., Alexander S. Zlobin, Olga O. Zaytseva, Lucija Klarić, Sodbo Z. Sharapov, Eugene D Pakhomov, Marcus Perola, et al. "Multivariate genome-wide analysis of immunoglobulin G N-glycosylation identifies new loci pleiotropic with immune function." Human Molecular Genetics 30, no. 13 (March 12, 2021): 1259–70. http://dx.doi.org/10.1093/hmg/ddab072.
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Abstract The N-glycosylation of immunoglobulin G (IgG) affects its structure and function. It has been demonstrated that IgG N-glycosylation patterns are inherited as complex quantitative traits. Genome-wide association studies identified loci harboring genes encoding enzymes directly involved in protein glycosylation as well as loci likely to be involved in regulation of glycosylation biochemical pathways. Many of these loci could be linked to immune functions and risk of inflammatory and autoimmune diseases. The aim of the present study was to discover and replicate new loci associated with IgG N-glycosylation and to investigate possible pleiotropic effects of these loci onto immune function and the risk of inflammatory and autoimmune diseases. We conducted a multivariate genome-wide association analysis of 23 IgG N-glycosylation traits measured in 8090 individuals of European ancestry. The discovery stage was followed up by replication in 3147 people and in silico functional analysis. Our study increased the total number of replicated loci from 22 to 29. For the discovered loci, we suggest a number of genes potentially involved in the control of IgG N-glycosylation. Among the new loci, two (near RNF168 and TNFRSF13B) were previously implicated in rare immune deficiencies and were associated with levels of circulating immunoglobulins. For one new locus (near AP5B1/OVOL1), we demonstrated a potential pleiotropic effect on the risk of asthma. Our findings underline an important link between IgG N-glycosylation and immune function and provide new clues to understanding their interplay.
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Majewska, Natalia I., Max L. Tejada, Michael J. Betenbaugh, and Nitin Agarwal. "N-Glycosylation of IgG and IgG-Like Recombinant Therapeutic Proteins: Why Is It Important and How Can We Control It?" Annual Review of Chemical and Biomolecular Engineering 11, no. 1 (June 7, 2020): 311–38. http://dx.doi.org/10.1146/annurev-chembioeng-102419-010001.
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Regulatory bodies worldwide consider N-glycosylation to be a critical quality attribute for immunoglobulin G (IgG) and IgG-like therapeutics. This consideration is due to the importance of posttranslational modifications in determining the efficacy, safety, and pharmacokinetic properties of biologics. Given its critical role in protein therapeutic production, we review N-glycosylation beginning with an overview of the myriad interactions of N-glycans with other biological factors. We examine the mechanism and drivers for N-glycosylation during biotherapeutic production and the several competing factors that impact glycan formation, including the abundance of precursor nucleotide sugars, transporters, glycosidases, glycosyltransferases, and process conditions. We explore the role of these factors with a focus on the analytical approaches used to characterize glycosylation and associated processes, followed by the current state of advanced glycosylation modeling techniques. This combination of disciplines allows for a deeper understanding of N-glycosylation and will lead to more rational glycan control.
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Su, Zhipeng, Qing Xie, Yanping Wang, and Yunsen Li. "Abberant Immunoglobulin G Glycosylation in Rheumatoid Arthritis by LTQ-ESI-MS." International Journal of Molecular Sciences 21, no. 6 (March 17, 2020): 2045. http://dx.doi.org/10.3390/ijms21062045.
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Aberrant glycosylation has been observed in many autoimmune diseases. For example, aberrant glycosylation of immunoglobulin G (IgG) has been implicated in rheumatoid arthritis (RA) pathogenesis. The aim of this study is to investigate IgG glycosylation and whether there is an association with rheumatoid factor levels in the serum of RA patients. We detected permethylated N-glycans of the IgG obtained in serum from 44 RA patients and 30 healthy controls using linear ion-trap electrospray ionization mass spectrometry (LTQ-ESI-MS), a highly sensitive and efficient approach in the detection and identification of N-glycans profiles. IgG N-glycosylation and rheumatoid factor levels were compared in healthy controls and RA patients. Our results suggested that total IgG purified from serum of RA patients shows significantly lower galactosylation (p = 0.0012), lower sialylation (p < 0.0001) and higher fucosylation (p = 0.0063) levels compared with healthy controls. We observed a positive correlation between aberrant N-glycosylation and rheumatoid factor level in the RA patients. In conclusion, we identified aberrant glycosylation of IgG in the serum of RA patients and its association with elevated levels of rheumatoid factor.
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Chinello, Clizia, Noortje de Haan, Giulia Capitoli, Barbara Trezzi, Antonella Radice, Lisa Pagani, Lucrezia Criscuolo, et al. "Definition of IgG Subclass-Specific Glycopatterns in Idiopathic Membranous Nephropathy: Aberrant IgG Glycoforms in Blood." International Journal of Molecular Sciences 23, no. 9 (April 23, 2022): 4664. http://dx.doi.org/10.3390/ijms23094664.
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The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) (n = 20), and 88 healthy control subjects. The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypo-galactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification.
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Greto, Valentina L., Ana Cvetko, Tamara Štambuk, Niall J. Dempster, Domagoj Kifer, Helena Deriš, Ana Cindrić, et al. "Extensive weight loss reduces glycan age by altering IgG N-glycosylation." International Journal of Obesity 45, no. 7 (May 3, 2021): 1521–31. http://dx.doi.org/10.1038/s41366-021-00816-3.
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Abstract Background Obesity, a major global health problem, is associated with increased cardiometabolic morbidity and mortality. Protein glycosylation is a frequent posttranslational modification, highly responsive to inflammation and ageing. The prospect of biological age reduction, by changing glycosylation patterns through metabolic intervention, opens many possibilities. We have investigated whether weight loss interventions affect inflammation- and ageing-associated IgG glycosylation changes, in a longitudinal cohort of bariatric surgery patients. To support potential findings, BMI-related glycosylation changes were monitored in a longitudinal twins cohort. Methods IgG N-glycans were chromatographically profiled in 37 obese patients, subjected to low-calorie diet, followed by bariatric surgery, across multiple timepoints. Similarly, plasma-derived IgG N-glycan traits were longitudinally monitored in 1680 participants from the TwinsUK cohort. Results Low-calorie diet induced a marked decrease in the levels of IgG N-glycans with bisecting GlcNAc, whose higher levels are usually associated with ageing and inflammatory conditions. Bariatric surgery resulted in extensive alterations of the IgG N-glycome that accompanied progressive weight loss during 1-year follow-up. We observed a significant increase in digalactosylated and sialylated glycans, and a substantial decrease in agalactosylated and core fucosylated IgG N-glycans (adjusted p value range 7.38 × 10−04–3.94 × 10−02). This IgG N-glycan profile is known to be associated with a younger biological age and reflects an enhanced anti-inflammatory IgG potential. Loss of BMI over a 20 year period in the TwinsUK cohort validated a weight loss-associated agalactosylation decrease (adjusted p value 1.79 × 10−02) and an increase in digalactosylation (adjusted p value 5.85 × 10−06). Conclusions Altogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced glycan and biological age.
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Štambuk, Jerko, Frano Vučković, Siniša Habazin, Maja Hanić, Mislav Novokmet, Susanna Nikolaus, Florian Tran, et al. "Distinct Longitudinal Changes in Immunoglobulin G N-Glycosylation Associate with Therapy Response in Chronic Inflammatory Diseases." International Journal of Molecular Sciences 23, no. 15 (July 30, 2022): 8473. http://dx.doi.org/10.3390/ijms23158473.
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Immunosuppressants and biologicals are widely used therapeutics for various chronic inflammatory diseases (CID). To gain more detailed insight into their downstream effects, we examined their impact on serum immunoglobulin G (IgG) glycosylation. We analyzed IgG subclass-specific fragment crystallizable (Fc) N-glycosylation in patients suffering from various CID using the LC-MS approach. Firstly, we compared IgG Fc N-glycosylation between 128 CID patients and 204 healthy controls. Our results replicated previously observed CID-related decrease in IgG Fc galactosylation (adjusted p-value range 1.70 × 10−2–5.95 × 10−22) and sialylation (adjusted p-value range 1.85 × 10−2–1.71 × 10−18). Secondly, to assess changes in IgG Fc N-glycosylation associated with therapy and remission status, we compared 139 CID patients receiving either azathioprine, infliximab, or vedolizumab therapy. We observed an increase in IgG Fc galactosylation (adjusted p-value range 1.98 × 10−2–1.30 × 10−15) and sialylation (adjusted p-value range 3.28 × 10−6–4.34 × 10−18) during the treatment. Furthermore, patients who reached remission displayed increased Fc galactosylation levels (p-value range 2.25 × 10−2–5.44 × 10−3) in comparison to patients with active disease. In conclusion, the alterations in IgG Fc glycosylation and the fact these changes are even more pronounced in patients who achieved remission, suggest modulation of IgG inflammatory potential associated with CID therapy.
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Suzuki, N. "Site-specific N-glycosylation of chicken serum IgG." Glycobiology 14, no. 3 (December 23, 2003): 275–92. http://dx.doi.org/10.1093/glycob/cwh031.
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Radovani, Barbara, Frano Vučković, Aldo P. Maggioni, Ele Ferrannini, Gordan Lauc, and Ivan Gudelj. "IgG N-Glycosylation Is Altered in Coronary Artery Disease." Biomolecules 13, no. 2 (February 16, 2023): 375. http://dx.doi.org/10.3390/biom13020375.
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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.
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Petit, Marie, Marie-Laure Walet-Balieu, Damien Schapman, Marie-Laure Golinski, Carole Burel, Marion Barray, Laurent Drouot, et al. "Longitudinal Pathogenic Properties and N-Glycosylation Profile of Antibodies from Patients with Pemphigus after Corticosteroid Treatment." Biomedicines 9, no. 10 (October 8, 2021): 1411. http://dx.doi.org/10.3390/biomedicines9101411.
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Pemphigus vulgaris is an autoimmune disease that occurs due to pathogenic autoantibodies that recognize the following epidermal adhesion proteins: desmogleins. Systemic corticosteroids usually decrease the titers of anti-desmoglein autoantibodies and improve patients’ conditions. Since modifications of IgG N-glycosylation have been described in some autoimmune diseases, we hypothesized that changes in the pathogenic activity of pemphigus IgG could be related to changes in their N-glycosylation profile. The purpose of this study was to assess, longitudinally, the pathogenicity of pemphigus serum IgG and their N-glycosylation profile during phases of disease activity and clinical remission. The pathogenic activity of serum IgG was measured in vitro on immortalized keratinocytes, by immunofluorescence and dissociation assays, and IgG N-glycans were analyzed by mass spectrometry. We showed (i) a correlation between pemphigus clinical activity and the pathogenicity of serum IgG at baseline and at month 6, while the persistence of the in vitro pathogenic activity of IgG during its evolution, even in patients in clinical remission, seemed to be predictive of relapse; (ii) that modifications of the N-glycan structure were altered the in vitro pathogenicity of patients’ autoantibodies; (iii) that the pathogenic properties of pemphigus IgG did not appear to be related to the disparity in IgG N-glycans during the course of pemphigus.
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Cvetko, Ana, Domagoj Kifer, Olga Gornik, Lucija Klarić, Elizabeth Visser, Gordan Lauc, James F. Wilson, and Tamara Štambuk. "Glycosylation Alterations in Multiple Sclerosis Show Increased Proinflammatory Potential." Biomedicines 8, no. 10 (October 13, 2020): 410. http://dx.doi.org/10.3390/biomedicines8100410.
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Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS), with unresolved aetiology. Previous studies have implicated N-glycosylation, a highly regulated enzymatic attachment of complex sugars to targeted proteins, in MS pathogenesis. We investigated individual variation in N-glycosylation of the total plasma proteome and of IgG in MS. Both plasma protein and IgG N-glycans were chromatographically profiled and quantified in 83 MS cases and 88 age- and sex-matched controls. Comparing levels of glycosylation features between MS cases and controls revealed that core fucosylation (p = 6.96 × 10−3) and abundance of high-mannose structures (p = 1.48 × 10−2) were the most prominently altered IgG glycosylation traits. Significant changes in plasma protein N-glycome composition were observed for antennary fucosylated, tri- and tetrasialylated, tri- and tetragalactosylated, high-branched N-glycans (p-value range 1.66 × 10−2–4.28 × 10−2). Classification performance of N-glycans was examined by ROC curve analysis, resulting in an AUC of 0.852 for the total plasma N-glycome and 0.798 for IgG N-glycome prediction models. Our results indicate that multiple aspects of protein glycosylation are altered in MS, showing increased proinflammatory potential. N-glycan alterations showed substantial value in classification of the disease status, nonetheless, additional studies are warranted to explore their exact role in MS development and utility as biomarkers.
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Ząbczyńska, Marta, Katarzyna Polak, Kamila Kozłowska, Grzegorz Sokołowski, and Ewa Pocheć. "The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto’s Thyroiditis: An in Vitro Model of Thyroid Autoimmunity." Biomolecules 10, no. 2 (January 22, 2020): 171. http://dx.doi.org/10.3390/biom10020171.
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Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are involved in destruction of thyroid tissue in Hashimoto’s thyroiditis (HT). N-glycosylation of the Fc fragment affects the effector functions of IgG by enhancing or suppressing the cytotoxicity effect. The aim of the present study was to assess the impact of HT-specific IgG glycosylation in ADCC and CDC, using in vitro models. The normal thyroid Nthy-ori 3-1 cell line and thyroid carcinoma FTC-133 cells were used as the target cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and the HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors and then treated with α2-3,6,8-neuraminidase to cut off sialic acids (SA) from N-glycans. We observed more intensive cytotoxicity in the presence of IgG from HT patients than in the presence of IgG from healthy donors. Removal of SA from IgG N-glycans increased ADCC intensity and reduced CDC. We conclude that the enhanced thyrocyte lysis resulted from the higher anti-TPO content in the whole IgG pool of HT donors and from altered IgG glycosylation in HT autoimmunity.
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Singh, Sunny S., Ralph Heijmans, Claudia K. E. Meulen, Aloysius G. Lieverse, Olga Gornik, Eric J. G. Sijbrands, Gordan Lauc, and Mandy van Hoek. "Association of the IgG N-glycome with the course of kidney function in type 2 diabetes." BMJ Open Diabetes Research & Care 8, no. 1 (April 2020): e001026. http://dx.doi.org/10.1136/bmjdrc-2019-001026.
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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 potential of IgG. We investigated the prospective relationship between IgG N-glycosylation patterns and kidney function in type 2 diabetes.Research design and methodsIn the DiaGene study, an all-lines-of-care case–control study (n=1886) with mean prospective follow-up of 7.0 years, the association between 58 IgG N-glycan profiles and estimated glomerular filtration rate (eGFR) and albumin-to-creatinine ratio (ACR) per year and during total follow-up was analyzed. Models were adjusted for clinical variables and multiple comparisons.ResultsEleven traits were significantly associated with eGFR change per year. Bisecting GlcNAc in fucosylated and fucosylated disialylated structures and monosialylation of fucosylated digalactosylated structures were associated with a faster decrease of eGFR. Fucosylation of neutral and monogalactosylated structures was associated with less eGFR decline per year. No significant associations between IgG glycans and ACR were found.ConclusionsIn type 2 diabetes, we found IgG N-glycosylation patterns associated with a faster decline of kidney function, reflecting a pro-inflammatory state of IgG. eGFR, but not ACR, was associated with IgG glycans, which suggests these associations may represent renal macroangiopathy rather than microvascular disease.
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Haslund-Gourley, Benjamin S., Brian Wigdahl, and Mary Ann Comunale. "IgG N-glycan Signatures as Potential Diagnostic and Prognostic Biomarkers." Diagnostics 13, no. 6 (March 7, 2023): 1016. http://dx.doi.org/10.3390/diagnostics13061016.
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IgG N-glycans are an emerging source of disease-specific biomarkers. Over the last decade, the continued development of glycomic databases and the evolution of glyco-analytic methods have resulted in increased throughput, resolution, and sensitivity. IgG N-glycans promote adaptive immune responses through antibody-dependent cellular cytotoxicity (ADCC) and complement activation to combat infection or cancer and promote autoimmunity. In addition to the functional assays, researchers are examining the ability of protein-specific glycosylation to serve as biomarkers of disease. This literature review demonstrates that IgG N-glycans can discriminate between healthy controls, autoimmune disease, infectious disease, and cancer with high sensitivity. The literature also indicates that the IgG glycosylation patterns vary across disease state, thereby supporting their role as specific biomarkers. In addition, IgG N-glycans can be collected longitudinally from patients to track treatment responses or predict disease reoccurrence. This review focuses on IgG N-glycan profiles applied as diagnostics, cohort discriminators, and prognostics. Recent successes, remaining challenges, and upcoming approaches are critically discussed.
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Simunovic, Jelena, Marija Vilaj, Irena Trbojevic-Akmacic, Ana Momcilovic, Frano Vuckovic, Ivan Gudelj, Julija Juric, Natali Nakic, Gordan Lauc, and Marija Pezer. "Comprehensive N-glycosylation analysis of immunoglobulin G from dried blood spots." Glycobiology 29, no. 12 (May 30, 2019): 817–21. http://dx.doi.org/10.1093/glycob/cwz061.
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Abstract Immunoglobulin G (IgG) glycans are emerging as a new putative biomarker for biological age and different diseases, requiring a robust workflow for IgG glycome analysis, ideally beginning with a simple and undemanding sampling procedure. Here, we report the first comprehensive study on total N-glycans of IgG isolated from dried blood spots (DBSs), which was performed in a high-throughput mode. We compared the IgG N-glycan profiles originating from DBS with those originating from plasma, compared different media for DBS collection, evaluated analytical variation and assessed IgG N-glycan profile stability for different storage conditions. In conclusion, we show that DBSs are a good and stable source material for a robust IgG N-glycan analysis by ultra-performance liquid chromatography, suitable for blood sampling in conditions where no trained personnel and necessary laboratory equipment are available.
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Lu, Lenette L., Jishnu Das, Patricia S. Grace, Sarah M. Fortune, Blanca I. Restrepo, and Galit Alter. "Antibody Fc Glycosylation Discriminates Between Latent and Active Tuberculosis." Journal of Infectious Diseases 222, no. 12 (February 15, 2020): 2093–102. http://dx.doi.org/10.1093/infdis/jiz643.
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Abstract Background Mycobacterium tuberculosis remains a global health problem and clinical management is complicated by difficulty in discriminating between latent infection and active disease. While M. tuberculosis-reactive antibody levels are heterogeneous, studies suggest that levels of IgG glycosylation differ between disease states. Here we extend this observation across antibody domains and M. tuberculosis specificities to define changes with the greatest resolving power. Methods Capillary electrophoretic glycan analysis was performed on bulk non-antigen–specific IgG, bulk Fc domain, bulk Fab domain, and purified protein derivative (PPD)- and Ag85A-specific IgG from subjects with latent (n = 10) and active (n = 20) tuberculosis. PPD-specific isotype/subclass, PPD-specific antibody-dependent phagocytosis, cellular cytotoxicity, and natural killer cell activation were assessed. Discriminatory potentials of antibody features were evaluated individually and by multivariate analysis. Results Parallel profiling of whole, Fc, and Fab domain-specific IgG glycosylation pointed to enhanced differential glycosylation on the Fc domain. Differential glycosylation was observed across antigen-specific antibody populations. Multivariate modeling highlighted Fc domain glycan species as the top discriminatory features, with combined PPD IgG titers and Fc domain glycans providing the highest classification accuracy. Conclusions Differential glycosylation occurs preferentially on the Fc domain, providing significant discriminatory power between different states of M. tuberculosis infection and disease.
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Gyebrovszki, Balázs, András Ács, Dániel Szabó, Felícia Auer, Soma Novozánszki, Bernadette Rojkovich, Anna Magyar, et al. "The Role of IgG Fc Region N-Glycosylation in the Pathomechanism of Rheumatoid Arthritis." International Journal of Molecular Sciences 23, no. 10 (May 23, 2022): 5828. http://dx.doi.org/10.3390/ijms23105828.
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Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)2 fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.
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Font, Guillaume, Marie-Laure Walet-Balieu, Marie Petit, Carole Burel, Maud Maho-Vaillant, Vivien Hébert, Philippe Chan, et al. "IgG N-Glycosylation from Patients with Pemphigus Treated with Rituximab." Biomedicines 10, no. 8 (July 22, 2022): 1774. http://dx.doi.org/10.3390/biomedicines10081774.
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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.
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Papakonstantinou, Maria, Georgios Dryllis, Maria Efstathopoulou, Dimitra Vlachopanou, Michalis Kechriotis, and Serena Valsami. "N-Glycosylation of IgG Immunoglobulin and its clinical significance." Journal of Biomedicine 4 (2019): 35–43. http://dx.doi.org/10.7150/jbm.33922.
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Sonneveld, Myrthe Elisabeth, Rosina Plomp, Jon Admiraal, Carolien Koeleman, Agnes Hipgrave-Ederveen, Joke Koelewijn, Peter Ligthart, et al. "IgG Alloantibodies Against RBC Induced By Pregnancy or Transfusion Have Unique Glycosylation Patterns Which Correlate with Clinical Outcome of Hemolytic Disease of the Fetus or Newborn." Blood 126, no. 23 (December 3, 2015): 660. http://dx.doi.org/10.1182/blood.v126.23.660.660.
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Hemolytic disease of the fetus or newborn (HDFN) is a potentially life-threatening disease in which red blood cells (RBC) of the child are destroyed by maternal IgG alloantibodies. HDFN caused by anti-D antibodies is well known, but may also occur due to other RBC antibody specificities, with anti-K, anti-c and anti-E being most frequently implicated (Koelewijn et al. Transfusion 2008;48(5):941-52). Besides alloantibody-mediated RBC destruction, anti-K also impairs erythropoiesis. The composition of the sugar moiety of the IgG-Fc tail influences the binding affinity to IgG Fc receptors (FcγR) on effector cells. Particularly, the absence of core fucose increases the binding of IgG to FcγRIIIa and FcγRIIIb. In previous studies we observed a correlation between fucosylation of anti-D and anti-platelet antibodies with severity of disease in patients with HDFN (n=8) or fetal or neonatal alloimmune thrombocytopenia (FNAIT) (n=48) (Kapur et al Br.J.Haematol. 2014 166 (6): 936-945; Kapur et al Blood 2014;123(4):471-80). The aim of this study was to investigate whether skewed antibody glycosylation also applies to other anti-RBC antibodies and if this is related to the immunization route and disease severity. Clinical data were collected for 146 women with anti-K, c or E antibodies. Anti-K (n=30), anti-c (n=69) and anti-E (n=47) antibodies were purified from serum by acid elution after incubation of the serum with matching positive RBC using antigen-negative RBC as negative-control for specificity of the procedure. Anti-RBC specific IgG antibodies and total IgG antibodies were subjected to tryptic digestion and resulting IgG1 Fc-derived glycopeptides analyzed by MALDI-TOF-Mass Spectrometry. Lowered fucosylation was observed for anti-K IgG (average 75.7% compared to 92.1% of total IgG, p<0.0001), but less than observed for anti-D IgG1 (52.29%, p=0.0008). Also fucosylation of anti-E IgG was lowered (91.1%, p=0.02), but significantly less pronounced then anti-K and anti-D. Surprisingly, anti-c fucosylation was not significantly skewed (89.9%, p=0.25), but with lowered fucose down to 60% in a few anti-c cases. Anti-E is in most cases naturally occurring, which was definitively proven in 17 cases without history of transfusion or E-positive pregnancy. Anti-K is in most cases induced by transfusion (Koelewijn et al BJOG 2009;116 (10): 1307-1314). Analyzing only cases of mothers with blood transfusion history and antigen negative fathers (n=15) also showed lowered fucosylation (p=0.014), suggesting that signatures of low IgG-fucosylation are not related to pregnancy responses. In 113 antigen-positive newborns at risk for HDFN (K: n=16, c: n=62, E: n=35), the glycosylation pattern of the specific IgG correlated with anaemia and severity of jaundice. For anti-K, galactosylation and sialylation correlated with hemoglobin (r=-0.78 ,p=0.0015, and r=-0.59, p=0.0303, resp.). Similar correlations were found with hematocrit levels. A low anti-c fucosylation correlated with increased bilirubin level (r=-0.38, p=0.0186) and severity of jaundice (r=-0.35, p=0.0103). Newborns exposed to anti-c that required a blood transfusion showed significantly lower galactosylation and higher bisection (p=0.03, p=0.01 resp.). In conclusion, we found similar skewing of antigen-specific IgG1 glycosylation for anti-E and anti-K as observed for anti-D and anti-platelet antibodies, although less pronounced. This skewing was not related to the immune response being induced in pregnancy, as similar features were also observed after transfusion. Interestingly, natural anti-E antibodies, probably evoked against non-blood borne antigens and recognizing E by molecular mimicry are far less skewed, possibly explaining why the presence of this antibody rarely has clinical consequences. The less pronounced skewing of glycosylation of anti-c might also explain the lower risk of severe HDFN mediated compared to anti-D. Anti-K glycosylation influenced clinical outcome, but in contrast to anti-Rh antibodies, it was not fucosylation but galactosylation and sialylation that correlated with anaemia. This might be related to the differences in the mechanism by which these antibodies induce anemia. Importantly, the clinical outcome of HDFN is strongly affected by IgG1-glycosylation, which is proving to become an important biomarker in screening for severe cases of HDFN. Disclosures No relevant conflicts of interest to declare.
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Blomme, Bram, Christophe Van Steenkiste, Paola Grassi, Stuart M. Haslam, Anne Dell, Nico Callewaert, and Hans Van Vlierberghe. "Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven." American Journal of Physiology-Gastrointestinal and Liver Physiology 300, no. 5 (May 2011): G833—G842. http://dx.doi.org/10.1152/ajpgi.00228.2010.
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N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl4 were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl4 models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl4 model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.
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Ruhaak, L. Renee, Cynthia Williams, Anne Fenton, Lauren Nagy, Qiuting Hong, Satya Dandekar, and Carlito Lebrilla. "Aberrant glycosylation of plasma proteins in HIV-infected patients (P6361)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 216.5. http://dx.doi.org/10.4049/jimmunol.190.supp.216.5.
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Abstract Background: Glycosylation is one of the most abundant post-translational modifications, and is involved in protein structure formation and protein-protein interactions. Aberrant glycosylation profiles are often observed on plasma proteins from patients with inflammatory diseases. Increased levels of non-galactosylated glycans have been reported on serum IgGs of HIV-infected patients. We investigated the effects of HIV infection on protein glycosylation by N-glycomic profiling of plasma and plasma IgG. Methods: We obtained plasma samples of 22 HIV infected patients (11 therapy-naïve, 11 receiving anti-retroviral therapy) and 11 HIV-negative controls. First, a nano-LC-TOF-MS strategy was employed for the evaluation of plasma N-glycan profiles in each of the samples. Then, a UPLC-QQQ-MS method was used to evaluate the IgG specific glycosylation patterns. N-glycan peak integrals were used for biostatistical analysis. Results and conclusion: Several neutral, fucosylated and sialylated glycan compositions as well as high mannose type glycans in plasma samples were significantly altered in therapy-naïve HIV infected patients compared to controls. Moreover, galactose-deficient glycans were increased on the IgG in these patients, independent of IgG subclass. Interestingly, these effects were largely reduced in HIV infected patients receiving therapy. These results suggest an important role for protein glycosylation in immune dysfunction that is driven by active HIV infection.
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Sołkiewicz, Katarzyna, Monika Kacperczyk, Hubert Krotkiewski, Marcin Jędryka, and Ewa Maria Kratz. "O-Glycosylation Changes in Serum Immunoglobulin G Are Associated with Inflammation Development in Advanced Endometriosis." International Journal of Molecular Sciences 23, no. 15 (July 22, 2022): 8087. http://dx.doi.org/10.3390/ijms23158087.
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Endometriosis is a gynecological disease, the pathogenesis of which seems to be directly related to inflammatory processes with an immune basis. Our study aimed to analyze the O-glycosylation of native serum IgG and IgG isolated from sera of women with advanced endometriosis, without endometriosis but with benign gynecological diseases, and from a control group of healthy women, in the context of its utility for differentiation of advanced endometriosis from the other two groups of women studied. For the analysis of serum IgG O-glycosylation and the expression of multi-antennary N-glycans, lectin-ELISA with lectins specific to O-glycans (MPL, VVL, and Jacalin) and highly branched N-glycans (PHA-L) was used. The relative reactivities of isolated serum IgG O-linked glycans with specific lectins as well as the MPL/VVL O-glycosylation ratio were significantly higher in patients with advanced endometriosis and those with other gynecological diseases when compared to the control group of healthy women. We also showed significantly higher expression of PHA-L-reactive multi-antennary N-glycans in isolated IgG in the advanced endometriosis and the non-endometriosis groups in comparison to the control group. Additionally, significantly higher expression of Jacalin-reactive O-glycans in isolated IgG was observed in the non-endometriosis than in the advanced endometriosis group. The results of the ROC curve and cluster analysis additionally confirmed that the lectin-based analysis of isolated serum IgG O-glycosylation and the expression of highly branched N-glycans may help distinguish women with advanced endometriosis from healthy women. Moreover, the analysis of the expression of Jacalin-reactive i-IgG O-glycans may be helpful in differentiation between women with advanced endometriosis and patients with other gynecological diseases with an inflammatory background. In the case of non-endometriosis patients, the observed differences were most probably caused by increased expression of core 3 type O-glycans.
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Larsen, Joachim Steen, Richard Torbjörn Gustav Karlsson, Weihua Tian, Morten Alder Schulz, Annemarie Matthes, Henrik Clausen, Bent Larsen Petersen, and Zhang Yang. "Engineering mammalian cells to produce plant-specific N-glycosylation on proteins." Glycobiology 30, no. 8 (February 10, 2020): 528–38. http://dx.doi.org/10.1093/glycob/cwaa009.
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Abstract Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and β2xylose (β2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/β2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.
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Klarić, Lucija, Yakov A. Tsepilov, Chloe M. Stanton, Massimo Mangino, Timo Tõnis Sikka, Tõnu Esko, Eugene Pakhomov, et al. "Glycosylation of immunoglobulin G is regulated by a large network of genes pleiotropic with inflammatory diseases." Science Advances 6, no. 8 (February 2020): eaax0301. http://dx.doi.org/10.1126/sciadv.aax0301.
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Effector functions of immunoglobulin G (IgG) are regulated by the composition of a glycan moiety, thus affecting activity of the immune system. Aberrant glycosylation of IgG has been observed in many diseases, but little is understood about the underlying mechanisms. We performed a genome-wide association study of IgG N-glycosylation (N = 8090) and, using a data-driven network approach, suggested how associated loci form a functional network. We confirmed in vitro that knockdown of IKZF1 decreases the expression of fucosyltransferase FUT8, resulting in increased levels of fucosylated glycans, and suggest that RUNX1 and RUNX3, together with SMARCB1, regulate expression of glycosyltransferase MGAT3. We also show that variants affecting the expression of genes involved in the regulation of glycoenzymes colocalize with variants affecting risk for inflammatory diseases. This study provides new evidence that variation in key transcription factors coupled with regulatory variation in glycogenes modifies IgG glycosylation and has influence on inflammatory diseases.
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Plavša, Branimir, Janko Szavits-Nossan, Aleksandar Blivajs, Borna Rapčan, Barbara Radovani, Igor Šesto, Krešimir Štambuk, et al. "The N-Glycosylation of Total Plasma Proteins and IgG in Atrial Fibrillation." Biomolecules 13, no. 4 (March 28, 2023): 605. http://dx.doi.org/10.3390/biom13040605.
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Atrial fibrillation is a disease with a complex pathophysiology, whose occurrence and persistence are caused not only by aberrant electrical signaling in the heart, but by the development of a susceptible heart substrate. These changes, such as the accumulation of adipose tissue and interstitial fibrosis, are characterized by the presence of inflammation. N-glycans have shown great promise as biomarkers in different diseases, specifically those involving inflammatory changes. To assess the changes in the N-glycosylation of the plasma proteins and IgG in atrial fibrillation, we analyzed the N-glycosylation of 172 patients with atrial fibrillation, before and six months after a pulmonary vein isolation procedure, with 54 cardiovascularly healthy controls. An analysis was performed using ultra-high-performance liquid chromatography. We found one oligomannose N-glycan structure from the plasma N-glycome and six IgG N-glycans, mainly revolving around the presence of bisecting N-acetylglucosamine, that were significantly different between the case and control groups. In addition, four plasma N-glycans, mostly oligomannose structures and a derived trait that was related to them, were found to be different in the patients who experienced an atrial fibrillation recurrence during the six-month follow-up. IgG N-glycosylation was extensively associated with the CHA2DS2-VASc score, confirming its previously reported associations with the conditions that make up the score. This is the first study looking at the N-glycosylation patterns in atrial fibrillation and warrants further investigation into the prospect of glycans as biomarkers for atrial fibrillation.
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Xue, Jing, Li-Ping Zhu, and Qiang Wei. "IgG-Fc N-glycosylation at Asn297 and IgA O-glycosylation in the hinge region in health and disease." Glycoconjugate Journal 30, no. 8 (June 20, 2013): 735–45. http://dx.doi.org/10.1007/s10719-013-9481-y.
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Maratha, Ashwini, Henning Stockmann, Karen P. Coss, M. Estela Rubio-Gozalbo, Ina Knerr, Maria Fitzgibbon, Terri P. McVeigh, et al. "Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis." European Journal of Human Genetics 24, no. 7 (January 6, 2016): 976–84. http://dx.doi.org/10.1038/ejhg.2015.254.
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Rombouts, Yoann, Annemiek Willemze, Joyce J. B. C. van Beers, Jing Shi, Priscilla F. Kerkman, Linda van Toorn, George M. C. Janssen, et al. "Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis." Annals of the Rheumatic Diseases 75, no. 3 (January 13, 2015): 578–85. http://dx.doi.org/10.1136/annrheumdis-2014-206598.
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ObjectivesTo understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from ‘conventional’ antibodies in rheumatoid arthritis (RA).MethodsSerum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications.ResultsIn all donor samples studied (n=24), ACPA-IgG exhibited a 10–20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens.ConclusionsThe vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the ‘germline-counterparts’ of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.
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Cao, Yedi, Zhijing Song, Yan Gong, Keli Zhao, Xue Zhao, Youyuan Huang, Chenxue Qu, et al. "The Immune Microenvironment of Hashimoto’s Thyroiditis Regulates the Glycosylation Modification of IgG." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A845. http://dx.doi.org/10.1210/jendso/bvab048.1724.
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Abstract Objectives: Elevation of anti-thyroglobulin antibodies that are primarily IgG isotype is a hallmark of Hashimoto’s thyroiditis (HT). As for IgG,it bears two conserved repertoire of N-linked glycans attached to its crystallizable fragment (Fc) at the 297 asparagine residue (Asn297). In our previous study, we found that serum TgAb IgG from HT patients exhibits higher glycosylation levels than those observed from healthy controls. Previous studies confirmed that imbalance of Th1/Th2 and Th17/Treg leading to altered immune microenvironment with elevation of certain cytokines was found in the thyroid tissue of HT, including IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF, APRIL. Thus, the aim of our study was to investigate the influence of the elevated cytokines on the differentiation process of B cells and the glycosylation levels of IgG. Methods: We formed a two-phase culture system in vitro to promote B cells to differentiate to antibody-secreting cells (ASCs). In the process of cell culture, B cells were co-cultured with cytokines as followed: IFN-γ, TNF-α, IL-21, IL-17A, IL-6, BAFF and APRIL. Flow cytometry was performed to identify the percentage of plasmablasts (CD38+CD27high) and plasma cells (CD20-CD138+). ELISA was used to measure the yield of IgG in culture supernatants. The glycosylation levels of secreted IgG under different stimulation conditions were detected by lectin microarray. Results: We found that IL-21, TNF-α and BAFF can significantly promote the differentiation of B cells into ASCs in vitro culture system, and augment the production of IgG to over 4-fold. In addition, cytokines affected the glycosylation modification profile of IgG diversely: 1) IL-21, IL-17A, TNF-α, BAFF significantly increased the glycosylation level of sialic acid of total IgG; 2) IFN-γ significantly increased the level of galactose; 3) IL-21, IL-17A, IFN-γ, BAFF, and APRIL significantly increased the level of mannose; 4) IL-6 significantly decreased the level of sialic acid, galactose and mannose; 5) IL-17A, IFN-γ, TNF-α, BAFF significantly increased the level of GalNAc that was a component of O-Glycan,which only exists in the hinge region of IgG3 subclass. Conclusions: The abnormally elevated cytokines in microenvironment participated in the regulation of B cell terminal differentiation process and glycosylation level of IgG, thereby involving in the pathogenesis of AITD.
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Tian, Cuihong, Gehendra Mahara, Hongxia Zhang, and Xuerui Tan. "Association of immunoglobulin G N-glycosylation with carotid atherosclerotic plaque phenotypes and actual clinical cardiovascular events: a study protocol for a longitudinal prospective cohort study." BMJ Open 12, no. 7 (July 2022): e058922. http://dx.doi.org/10.1136/bmjopen-2021-058922.
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IntroductionImmune-inflammatory response plays a key role in the pathogenesis of atherosclerosis. IgG N-glycosylation is reported to be associated with the 10-year atherosclerotic cardiovascular disease risk score and subclinical atherosclerosis. However, the relationship of IgG glycosylation with actual clinical cardiovascular disease (CVD) events and plaque phenotypes has rarely been investigated. Therefore, this study aims to understand whether IgG glycosylation traits are correlated with actual clinical CVD events and plaque phenotypes.Methods and analysisDesigned to verify the efficacy of IgG glycosylation as a risk for CVD events and screen potential biomarkers of CVD to prevent atherosclerosis occurrence, this longitudinal prospective cohort study will be conducted at the First Affiliated Hospital of Shantou University Medical College, China. In total, 2720 participants routinely examined by carotid ultrasound will be divided into different groups according to plaque phenotype characteristics. Ultra-performance liquid chromatography will be performed to separate and detect IgG N-glycans in serum collected at baseline and at the end of the first, second and third years. The primary outcome is the actual clinical CVD composite events, including non-fatal myocardial infarction, death due to coronary heart disease, and fatal or non-fatal stroke.Ethics and disseminationThe Clinical Ethics Committee of the First Affiliated Hospital of Shantou University Medical College approved this study (number: B-2021-127). Findings of this study will be submitted for publication in peer-reviewed journals.Trial registration numberChiCTR2100048740.
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Hipgrave Ederveen, Agnes L., Noortje de Haan, Melissa Baerenfaenger, Dirk J. Lefeber, and Manfred Wuhrer. "Dissecting Total Plasma and Protein-Specific Glycosylation Profiles in Congenital Disorders of Glycosylation." International Journal of Molecular Sciences 21, no. 20 (October 15, 2020): 7635. http://dx.doi.org/10.3390/ijms21207635.
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Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.
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Kissel, Theresa, Karin Anna van Schie, Lise Hafkenscheid, Anders Lundquist, Heidi Kokkonen, Manfred Wuhrer, Tom WJ Huizinga, Hans Ulrich Scherer, René Toes, and Solbritt Rantapää-Dahlqvist. "On the presence of HLA-SE alleles and ACPA-IgG variable domain glycosylation in the phase preceding the development of rheumatoid arthritis." Annals of the Rheumatic Diseases 78, no. 12 (August 30, 2019): 1616–20. http://dx.doi.org/10.1136/annrheumdis-2019-215698.
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ObjectiveAnti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients display a unique feature defined by the abundant presence of N-linked glycans within the variable domains (V-domains). Recently, we showed that N-glycosylation sites, which are required for the incorporation of V-domain glycans, are introduced following somatic hypermutation. However, it is currently unclear when V-domain glycosylation occurs. Further, it is unknown which factors might trigger the generation of V-domain glycans and whether such glycans are relevant for the transition towards RA. Here, we determined the presence of ACPA-IgG V-domain glycans in paired samples of pre-symptomatic individuals and RA patients.MethodsACPA-IgG V-domain glycosylation was analysed using ultra-high performance liquid chromatography (UHPLC) in paired samples of pre-symptomatic individuals (median interquartile range (IQR) pre-dating time: 5.8 (5.9) years; n=201; 139 ACPA-positive and 62 ACPA-negative) and RA patients (n=99; 94 ACPA-positive and 5 ACPA-negative).ResultsV-domain glycans on ACPA-IgG were already present up to 15 years before disease in pre-symptomatic individuals and their abundance increased closer to symptom onset. Noteworthy, human leucocyte antigen class II shared epitope (HLA-SE) alleles associated with the presence of V-domain glycans on ACPA-IgG.ConclusionOur observations indicate that somatic hypermutation of ACPA, which results in the incorporation of N-linked glycosylation sites and consequently V-domain glycans, occurs already years before symptom onset in individuals that will develop RA later in life. Moreover, our findings provide first evidence that HLA-SE alleles associate with ACPA-IgG V-domain glycosylation in the pre-disease phase and thereby further refine the connection between HLA-SE and the development of ACPA-positive RA.
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Cobb, Brian A. "The history of IgG glycosylation and where we are now." Glycobiology 30, no. 4 (August 27, 2019): 202–13. http://dx.doi.org/10.1093/glycob/cwz065.
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Abstract IgG glycosylation is currently at the forefront of both immunology and glycobiology, likely due in part to the widespread and growing use of antibodies as drugs. For over four decades, it has been recognized that the conserved N-linked glycan on asparagine 297 found within the second Ig domain of the heavy chain (CH2) that helps to comprise Fc region of IgG plays a special role in IgG structure and function. Changes in galactosylation, fucosylation and sialylation are now well-established factors, which drive differential IgG function, ranging from inhibitory/anti-inflammatory to activating complement and promoting antibody-dependent cellular cytotoxicity. Thus, if we are to truly understand how to design and deploy antibody-based drugs with maximal efficacy and evaluate proper vaccine responses from a protective and functional perspective, a deep understanding of IgG glycosylation is essential. This article is intended to provide a comprehensive review of the IgG glycosylation field and the impact glycans have on IgG function, beginning with the earliest findings over 40 years ago, in order to provide a robust foundation for moving forward.
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Wu, Zhiyuan, Huiying Pan, Di Liu, Di Zhou, Lixin Tao, Jie Zhang, Xiaonan Wang, et al. "Variation of IgG N ‐linked glycosylation profile in diabetic retinopathy." Journal of Diabetes 13, no. 8 (February 18, 2021): 672–80. http://dx.doi.org/10.1111/1753-0407.13160.
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Quast, Isaak, Benjamin Peschke, and Jan D. Lünemann. "Regulation of antibody effector functions through IgG Fc N-glycosylation." Cellular and Molecular Life Sciences 74, no. 5 (September 17, 2016): 837–47. http://dx.doi.org/10.1007/s00018-016-2366-z.
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Hutter, Sandro, Moritz Wolf, Nan Papili Gao, Dario Lepori, Thea Schweigler, Massimo Morbidelli, and Rudiyanto Gunawan. "Glycosylation Flux Analysis of Immunoglobulin G in Chinese Hamster Ovary Perfusion Cell Culture." Processes 6, no. 10 (October 1, 2018): 176. http://dx.doi.org/10.3390/pr6100176.
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The terminal sugar molecules of the N-linked glycan attached to the fragment crystalizable (Fc) region is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) such as immunoglobulin G (IgG). There exists naturally-occurring heterogeneity in the N-linked glycan structure of mAbs, and such heterogeneity has a significant influence on the clinical safety and efficacy of mAb drugs. We previously proposed a constraint-based modeling method called glycosylation flux analysis (GFA) to characterize the rates (fluxes) of intracellular glycosylation reactions. One contribution of this work is a significant improvement in the computational efficiency of the GFA, which is beneficial for analyzing large datasets. Another contribution of our study is the analysis of IgG glycosylation in continuous perfusion Chinese Hamster Ovary (CHO) cell cultures. The GFA of the perfusion cell culture data indicated that the dynamical changes of IgG glycan heterogeneity are mostly attributed to alterations in the galactosylation flux activity. By using a random forest regression analysis of the IgG galactosylation flux activity, we were further able to link the dynamics of galactosylation with two process parameters: cell-specific productivity of IgG and extracellular ammonia concentration. The characteristics of IgG galactosylation dynamics agree well with what we previously reported for fed-batch cultivations of the same CHO cell strain.
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SOTILLO, JAVIER, ALBA CORTÉS, CARLA MUÑOZ-ANTOLI, BERNARD FRIED, J. GUILLERMO ESTEBAN, and RAFAEL TOLEDO. "The effect of glycosylation of antigens on the antibody responses againstEchinostoma caproni(Trematoda: Echinostomatidae)." Parasitology 141, no. 10 (May 14, 2014): 1333–40. http://dx.doi.org/10.1017/s0031182014000596.
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SUMMARYIn the present study, we analyse the effect of glycosylation inEchinostoma caproni(Trematoda: Echinostomatidae) antigens in antibody responses against the parasite in experimentally infected mice. It has been previously demonstrated that the mouse is a host of high compatibility withE. caproniand develops elevated responses of IgG, IgG1, IgG3 and IgM as a consequence of the infection, though the role of glycans in these responses remains unknown. To this purpose, the responses generated in mice against non-treated excretory/secretory antigens ofE. caproniwere compared with those observed after N-deglycosylation, O-deglycosylation and double deglycosylation of the antigens by indirect ELISA and western blot. Our results suggest thatE. caproni-expressed glycans play a major role in the modulation of the immune responses. The results obtained indicate that IgG subclass responses generated in mice againstE. caproniare essentially due to glycoproteins and may affect the Th1/Th2 biasing. The reactivity significantly decreased after any of the deglycosylation treatments and the N-glycans appears to be of greater importance than O-glycans. Interestingly, the IgM response increased after N-deglycosylation suggesting that carbohydrates may mask peptide antigens.
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Shade, Kai-Ting C., Barbara Platzer, Nathaniel Washburn, Vinidhra Mani, Yannic C. Bartsch, Michelle Conroy, Jose D. Pagan, et al. "A single glycan on IgE is indispensable for initiation of anaphylaxis." Journal of Experimental Medicine 212, no. 4 (March 30, 2015): 457–67. http://dx.doi.org/10.1084/jem.20142182.
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Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell–bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.
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Forest-Nault, Catherine, Jimmy Gaudreault, Olivier Henry, Yves Durocher, and Gregory De Crescenzo. "On the Use of Surface Plasmon Resonance Biosensing to Understand IgG-FcγR Interactions." International Journal of Molecular Sciences 22, no. 12 (June 21, 2021): 6616. http://dx.doi.org/10.3390/ijms22126616.
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Surface plasmon resonance (SPR)-based optical biosensors offer real-time and label-free analysis of protein interactions, which has extensively contributed to the discovery and development of therapeutic monoclonal antibodies (mAbs). As the biopharmaceutical market for these biologics and their biosimilars is rapidly growing, the role of SPR biosensors in drug discovery and quality assessment is becoming increasingly prominent. One of the critical quality attributes of mAbs is the N-glycosylation of their Fc region. Other than providing stability to the antibody, the Fc N-glycosylation influences immunoglobulin G (IgG) interactions with the Fcγ receptors (FcγRs), modulating the immune response. Over the past two decades, several studies have relied on SPR-based assays to characterize the influence of N-glycosylation upon the IgG-FcγR interactions. While these studies have unveiled key information, many conclusions are still debated in the literature. These discrepancies can be, in part, attributed to the design of the reported SPR-based assays as well as the methodology applied to SPR data analysis. In fact, the SPR biosensor best practices have evolved over the years, and several biases have been pointed out in the development of experimental SPR protocols. In parallel, newly developed algorithms and data analysis methods now allow taking into consideration complex biomolecular kinetics. In this review, we detail the use of different SPR biosensing approaches for characterizing the IgG-FcγR interactions, highlighting their merit and inherent experimental complexity. Furthermore, we review the latest SPR-derived conclusions on the influence of the N-glycosylation upon the IgG-FcγR interactions and underline the differences and similarities across the literature. Finally, we explore new avenues taking advantage of novel computational analysis of SPR results as well as the latest strategies to control the glycoprofile of mAbs during production, which could lead to a better understanding and modelling of the IgG-FcγRs interactions.
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Mittermayr, Stefan, Jonathan Bones, Giao N. Le, and Peter O'Gorman. "N-Glycan Analysis of Polyclonal IgG from Patients with Multiple Myeloma Enables Classification of Stage Specific Pathologies." Blood 126, no. 23 (December 3, 2015): 1761. http://dx.doi.org/10.1182/blood.v126.23.1761.1761.
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Abstract Background Multiple myeloma (MM) is an incurable plasma cell malignancy, with eventual disease refractory and relapse. Its benign precursor, monoclonal gammopathy of undetermined significance (MGUS), has an annual transformation rate of 1%, while that of the asymptomatic smouldering myeloma (SMM) is 10%. The pathognomonic feature is the presence abnormal monoclonal immunoglobulin, of which immunoglobulin G (IgG) paraprotein is the most common. All subclasses of Igs are post-translationally modified by the addition of N-glycans, reportedly influencing structure, stability, and biological function. Previous studies in MM IgG had suggested an increase in the glycosylation of the antigen-binding fragment (Fab), and an overall elevation of sialylation. Using glycomic platforms, we aimed to investigate and characterise the IgG N-glycosylation profiles across the myeloma disease spectrum. Methods Polyclonal IgG was extracted from sera of patients with MM, SMM, MGUS, and age-matched control, using Protein G affinity chromatography. The quantity of extracted IgG was determined using the Bradford assay. N-glycans were enzymatically liberated from a normalised quantity of purified IgG, fluorescently labelled and profiled using hydrophilic interaction ultra-performance liquid chromatography. A combination of exoglycosidase digestions and mass spectrometry were used to elucidate the glycan structures with full linkage and positional specificity. Localisation analyses were performed to determine the distribution of N-glycans at asparagine 297 in the Fc region of the antibody and those present at any additional glycosylation sites present in the Fab region using a combination of enzymatic digestion using the commercially available IdeS enzyme and chemical reduction followed by SDS-PAGE separation of the resulting protein fragments and subsequent in-gel digestion of the N-glycans. Non-supervised principal component analysis (PCA) was employed to detect distinguishable chromatographic features among the studied groups. Longitudinal analyses of samples from individual patients collected during multiple clinical assessments were also performed. Results N-glycan analysis of polyclonal IgG showed unique N-glycan peaks with statistically significant chromatogram variation across the 4 studied groups. PCA identified specific patterns of glycosylation present in the glycan profiles, thus demonstrating the ability to distinguish between MGUS, SMM, MM and control. Sialylated biantennary N-glycans and N-glycans containing bisecting GlcNAc residues contributed most to the PCA separation. Further characterisation of the glycans using sialic acid linkage specific derivatisation and LC-MS analysis confirmed that sialic acids were present in an α2-6 linked configuration. Localisation analysis revealed N-glycans present in the Fc region of the extracted polyclonal antibodies consisted of the standard biantennary type glycans with core fucosylation, variable degrees of galactosylation and low levels of sialylation. Such oligosaccharide structures suggest that the Fc regions of polyclonal IgG present in patients with varying stages of plasma cell disorder maintain a correct orientation to facilitate interaction with Fcγ receptors. Sialylated biantennary N-glycans, identified during global polyclonal IgG glycosylation profiling, were found to be located predominantly in the Fab region of the antibody. The formation of these larger highly sialylated N-glycan structures is likely due to the removal of steric hindrance resulting in more facilitated access by the associated glycosyltransferases required for oligosaccharide biosynthesis. The presence of these charged oligosaccharide structures, with their inherent structural dynamics, are likely to affect the ability of the antibody to recognise antigen and form an immune complex. Conclusions Glycan analysis of polyclonal IgG extracted from the sera of patients with varying stages of myeloma progression is reported. Differential glycosylation on polyclonal IgG was observed between the patients with MGUS, SMM, and MM, with alterations in the levels of sialylated biantennary structures and glycans bearing bisecting GlcNAc residues capable of distinguishing between the patient groups in the spectrum of plasma cell disorders. Disclosures No relevant conflicts of interest to declare.
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Tsuchiya, N., T. Endo, K. Matsuta, S. Yoshinoya, F. Takeuchi, Y. Nagano, M. Shiota, K. Furukawa, N. Kochibe, and K. Ito. "Detection of glycosylation abnormality in rheumatoid IgG using N-acetylglucosamine-specific Psathyrella velutina lectin." Journal of Immunology 151, no. 2 (July 15, 1993): 1137–46. http://dx.doi.org/10.4049/jimmunol.151.2.1137.
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Abstract Although the galactose deficiency in the Asn297-linked sugar chains of serum IgG from patients with rheumatoid arthritis (RA) has been established, structural analysis of sugar chains has not been readily available. Psathyrella velutina lectin (PVL) preferentially interacts with the N-acetylglucosamine beta 1-->2Man group, exposed at the termini of sugar chains in agalacto IgG. Biotinylated PVL reacted strongly in Western blotting with H chains of IgG derived from patients with RA. An ELISA-based assay for the detection of agalacto IgG was developed using recombinant protein G and biotinylated PVL in combination, and the screening of patients' sera was performed. PVL binding of serum IgG significantly correlated with percentage of galactose-deficient IgG determined by the structural analysis. Age-related slight increase in PVL binding was observed among normal controls. Patients with RA showed significantly higher PVL binding (37.90 +/- 42.25 U/ml, n = 93) as compared with normal controls (5.75 +/- 2.92 U/ml, n = 112) (p = 0.0001). Patients with SLE showed lower but still significant PVL binding (17.86 +/- 5.18 U/ml, n = 10, p = 0.0001). PVL binding correlated with C-reactive protein level in serial analysis of individual RA patients, and was significantly higher in the synovial fluid compared with paired serum samples. PVL binding assay may provide an ideal tool for the simple and sensitive detection of agalacto IgG.
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ERCAN, Altan. "Sex effect on the correlation of immunoglobulin G glycosylation with rheumatoid arthritis disease activity." TURKISH JOURNAL OF BIOLOGY 44, no. 6 (December 14, 2020): 406–16. http://dx.doi.org/10.3906/biy-2005-7.
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Rheumatoid arthritis (RA) is a chronic autoimmune disease which affects females more than males with a presence of auto-antibodies. Immunoglobulin G (IgG) produced by adaptive arm has 2 functional domains, Fc and Fab. The Fc domain binds Fc gamma receptors and C1q proteins of the innate arm. Therefore, the IgG Fc domain serves as a bridge between the innate and adaptive arms and is regulated by an evolutionarily conserved N-glycosylation with variable structures. These glycans are classified as agalactosylated G0, monogalactosylated G1, and digalactosylated G2, which are further modified by core-fucosylation (F) and bisecting N-acetylglu-cosamine (B) moieties such as G0F and G0FB. Interestingly, proinflammatory G0F is shown to be regulated by estrogen in vivo. Here, it is hypothesized that the regulation of G0F by estrogen contributes to sex dichotomy in RA by setting up the level of IgG-dependent inflammation and therefore, RA disease activity (Das28-CRP3). To investigate this hypothesis, IgG glycosylation was characterized in serum samples from active RA patients (n = 232) and healthy controls (n = 232) by serum N-glycan analysis using the high performance liquid chromatography. According to the results, the IgG Fc glycan phenotype originates predominantly from the structure of G0F, and both G0F and G0FB correlate with Das28-CRP3 in females, but not in males. In conclusion, IgG G0F-dependent inflammation differs in males and females, and these differences point to the differential regulation of inflammation by sex hormone estrogen via IgG gly-cosylation.
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Lee, Ju Yeon, Jin-Woong Choi, Seoyoung Hwang, Sung Ho Hahm, and Yeong Hee Ahn. "Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping." International Journal of Molecular Sciences 23, no. 19 (October 5, 2022): 11807. http://dx.doi.org/10.3390/ijms231911807.
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The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.
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Field, M. C., S. Amatayakul-Chantler, T. W. Rademacher, P. M. Rudd, and R. A. Dwek. "Structural analysis of the N-glycans from human immunoglobulin A1: comparison of normal human serum immunoglobulin A1 with that isolated from patients with rheumatoid arthritis." Biochemical Journal 299, no. 1 (April 1, 1994): 261–75. http://dx.doi.org/10.1042/bj2990261.
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The primary structures of the N-linked oligosaccharides from normal human serum IgA1 were determined by a combination of sequential exoglycosidase digestion, Bio-Gel P-4 chromatography, anion-exchange chromatography and one-dimensional n.m.r. spectroscopy. Three major N-linked disialylated biantennary-complex-type structures were found (55%). The remaining N-linked oligosaccharides consisted of at least nine further structures, some of which (7%) were of the triantennary type and included disialylated triantennary oligosaccharides with outer-arm fucose substitution [Fuc alpha 1-3(4)]. Compared with IgG, the N-glycan structures on IgA are more completely processed: the outer arms have a higher proportion of galactose and sialic acid, and only trace levels of incompletely galactosylated oligosaccharides, commonly found on IgG, were detected. Analysis of the sialylated O-glycans revealed that 64% were [NeuAc2 alpha 3(6)]2Gal beta 3GalNAc and 9% were [NeuAc2 alpha 3(6)]-Gal beta 4GlcNAc beta 6[NeuAc2 alpha 3(6)Gal beta 3]GalNAc, and 27% were monosialylated. The N-linked glycosylation of both serum IgA1 and IgG isolated from a group of six normal individuals was compared with that from ten patients with rheumatoid arthritis (RA). In contrast with the hypogalactosylation found in IgG from diseased sera, there was no evidence of an equivalent decrease in the galactosylation of the IgA1 oligosaccharides. In addition, the N-glycosylation of IgA1 was remarkably consistent within the group of normal individuals. These data suggest that incomplete galactosylation of N-linked glycans and its augmentation in RA does not extend to IgA1 and that the RA-associated galactosyltransferase deficiency may be restricted to cells producing gamma-chain.
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Westhrin, Marita, Vlado Kovcic, Albert Bondt, Stephanie Holst, Zejian Zhang, Anders Sundan, Tobias S. Slørdahl, Anders Waage, Manfred Wuhrer, and Therese Standal. "Glycosylationof Immunoglobulins Determine Bone Loss in Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 4324. http://dx.doi.org/10.1182/blood-2019-122542.
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About 80% of patients with multiple myeloma develop a severe osteolytic bone disease causing pain and fractures. The myeloma cells secrete immunoglobulins and the presence of monoclonal immunoglobulins in serum is a hallmark of the disease. Immunoglobulins play a role in bone loss, but have not been linked with the bone disease in multiple myeloma. In this work, we isolated immunoglobulins from serum of myeloma patients using protein G coupled magnetic beads and columns. We found that immunoglobulins from patients with bone disease (n=16) promoted osteoclast differentiation when added to human monocyte-derived pre-osteoclasts (p=0.006). We next fractionated the immunoglobulin samples by size-exclusion chromatography and found that the "osteoclast promoting activity" was in the high-molecular weight fractions, suggesting that they are in complexes. Since extent of complex formation may be determined by glycosylation, we examined whether there is a difference in immunoglobulin glycosylation between healthy controls and patients, and whether it changes during disease progression. To this end we analysed IgG glycosylation in serum samples from patients (n=72) and age and sex matched controls (n=51). These analyses showed that patient IgG was less galactosylated (p=0.02) and less sialylated (p=0.04) compared with control IgG. Moreover, patients with bone disease (n=43) had significantly less galactose on IgG compared with patients without bone disease (p=0.02, n=33). Supporting this data, we found that galactosidase treatment of immunoglobulins from patients without bone disease induced osteoclastogenesis (p=0.03), whereas addition of galactose to immunoglobulins of patients with bone disease removed their pro-osteoclastogenic effect (p=0.01). Further, the glycosyltransferases ST6GAL1 and B4GALT11, which add sialic acid and galactose to the sugar chain, respectively, are less expressed in plasma cells obtained from patients with bone disease (n=137) compared with those without (n=36, p<0.002, p<0.001, GSE755). Importantly, we observed a significant reduction of IgG glycosylation (p=0.02, n=8) in serum samples obtained from individual patients before and after the onset of bone disease. Taken together, our data support that immunoglobulins promote bone loss in multiple myeloma. Disclosures No relevant conflicts of interest to declare.
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Meng, Xiaoni, Manshu Song, Marija Vilaj, Jerko Štambuk, Mamatyusupu Dolikun, Jie Zhang, Di Liu, et al. "Glycosylation of IgG Associates with Hypertension and Type 2 Diabetes Mellitus Comorbidity in the Chinese Muslim Ethnic Minorities and the Han Chinese." Journal of Personalized Medicine 11, no. 7 (June 29, 2021): 614. http://dx.doi.org/10.3390/jpm11070614.
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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.