IgG N-glycans Research Articles

Immunoglobulin G N-glycan Biomarkers for Autoimmune Diseases: Current State and a Glycoinformatics Perspective.

The effective treatment of autoimmune disorders can greatly benefit from disease-specific biomarkers that are functionally involved in immune system regulation and can be collected through minimally invasive procedures. In this regard, human serum IgG N-glycans are promising for uncovering disease predisposition and monitoring progression, and for the identification of specific molecular targets for advanced therapies. In particular, the IgG N-glycome in diseased tissues is considered to be disease-dependent; thus, specific glycan structures may be involved in the pathophysiology of autoimmune diseases. This study provides a critical overview of the literature on human IgG N-glycomics, with a focus on the identification of disease-specific glycan alterations. In order to expedite the establishment of clinically-relevant N-glycan biomarkers, the employment of advanced computational tools for the interpretation of clinical data and their relationship with the underlying molecular mechanisms may be critical. Glycoinformatics tools, including artificial intelligence and systems glycobiology approaches, are reviewed for their potential to provide insight into patient stratification and disease etiology. Challenges in the integration of such glycoinformatics approaches in N-glycan biomarker research are critically discussed.

Changes in Specific Biomarkers Indicate Cardiac Adaptive and Anti-inflammatory Response of Repeated Recreational SCUBA Diving.

ObjectiveRecreational SCUBA (rSCUBA) diving has become a highly popular and widespread sport. Yet, information on molecular events underlying (patho)physiological events that follow exposure to the specific environmental conditions (hyperbaric conditions, coldness, immersion, and elevated breathing pressure), in which rSCUBA diving is performed, remain largely unknown. Our previous study suggested that repeated rSCUBA diving triggers an adaptive response of cardiovascular and immune system. To elucidate further molecular events underlying cardiac and immune system adaptation and to exclude possible adverse effects we measured blood levels of specific cardiac and inflammation markers.MethodsThis longitudinal intervention study included fourteen recreational divers who performed five dives, one per week, on the depth 20–30 m that lasted 30 min, after the non-dive period of 5 months. Blood samples were taken immediately before and after the first, third, and fifth dives. Copeptin, immunoglobulins A, G and M, complement components C3 and C4, and differential blood count parameters, including neutrophil-to-lymphocyte ratio (NLR) were determined using standard laboratory methods. Cell-free DNA was measured by qPCR analysis and N-glycans released from IgG and total plasma proteins (TPP), were analyzed by hydrophilic interaction ultra-performance liquid chromatography.ResultsCopeptin level increased after the first dive but decreased after the third and fifth dive. Increases in immunoglobulins level after every dive and during whole studied period were observed, but no changes in C3, C4, and cfDNA level were detected. NLR increased only after the first dive. IgG and TPP N-glycosylation alterations toward anti-inflammatory status over whole studied period were manifested as an increase in monogalyctosylated and core-fucosylated IgG N-glycans and decrease in agalactosylated TPP N-glycans.ConclusionrSCUBA diving practiced on a regular basis promotes anti-inflammatory status thus contributing cardioprotection and conferring multiple health benefits.

N-Glycosylation Patterns across the Age-Related Macular Degeneration Spectrum.

The pathogenesis of age-related macular degeneration (AMD) remains elusive, despite numerous research studies. Therefore, we aimed to investigate the changes of plasma and IgG-specific N-glycosylation across the disease severity spectrum. We examined 2835 subjects from the 10.001 Dalmatians project, originating from the isolated Croatian islands of Vis and Korčula. All subjects were classified into four groups, namely (i) bilateral AMD, (ii) unilateral AMD, (iii) early-onset drusen, and (iv) controls. We analysed plasma and IgG N-glycans measured by HPLC and their association with retinal fundus photographs. There were 106 (3.7%) detected cases of AMD; 66 of them were bilateral. In addition, 45 (0.9%) subjects were recorded as having early-onset retinal drusen. We detected several interesting differences across the analysed groups, suggesting that N-glycans can be used as a biomarker for AMD. Multivariate analysis suggested a significant decrease in the immunomodulatory bi-antennary glycan structures in unilateral AMD (adjusted odds ratio 0.43 (95% confidence interval 0.22–0.79)). We also detected a substantial increase in the pro-inflammatory tetra-antennary plasma glycans in bilateral AMD (7.90 (2.94–20.95)). Notably, some of these associations were not identified in the aggregated analysis, where all three disease stages were collapsed into a single category, suggesting the need for better-refined phenotypes and the use of disease severity stages in the analysis of more complex diseases. Age-related macular degeneration progression is characterised by the complex interplay of various mechanisms, some of which can be detected by measuring plasma and IgG N-glycans. As opposed to a simple case-control study, more advanced and refined study designs are needed to understand the pathogenesis of complex diseases.

N‐glycan profiling alterations of serum and immunoglobulin G in immune thrombocytopenia

BackgroundThe glycosylation alterations of serum and IgG are involved in a variety of autoimmune and inflammatory diseases and have shown great potential in biomarker field. The diagnosis of immune thrombocytopenia (ITP) is exclusive. Our study aimed to discover the potential glyco‐biomarkers for auxiliary diagnosis of ITP.MethodsThe serum samples were obtained from 61 ITP patients and 35 healthy controls, and IgG samples were purified from 34 out of 61 ITP patients and 35 healthy controls. DNA sequencer‐assisted fluorophore‐assisted carbohydrate electrophoresis (DSA‐FACE) was used to analyze serum and IgG N‐glycan profiling.Results6 of 12 serum N‐glycan peaks, 6 of 7 IgG N‐glycan peaks, serum fucosylation, and IgG galactosylation were significantly different between ITP patients and healthy controls (p < 0.05). IgG peak 7 showed good diagnostic efficacy for discriminating ITP patients from healthy individuals (AUC 0.967). ITP patients with severe thrombocytopenia had a significantly lower serum fucosylation than ITP patients with mild and moderate thrombocytopenia (p < 0.05). Serum fucosylation and serum peak 5 were correlated with platelet counts in ITP patients with severe thrombocytopenia, and the absolute values of correlation coefficient were both over 0.5.ConclusionsThe specific N‐glycan patterns of serum and IgG were observed in ITP patients. IgG peak 7 was a potential biomarker for auxiliary diagnosis of ITP.

Longitudinal Pathogenic Properties and N-Glycosylation Profile of Antibodies from Patients with Pemphigus after Corticosteroid Treatment.

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.

Diagnostic Potential of Plasma IgG N-glycans in Discriminating Thyroid Cancer from Benign Thyroid Nodules and Healthy Controls.

BackgroundNovel biomarkers are urgently needed to distinguish between benign and malignant thyroid nodules and detect thyroid cancer in the early stage. The associations between serum IgG N-glycosylation and thyroid cancer risk have been revealed. We aimed to explore the potential of IgG N-glycan traits as biomarkers in the differential diagnosis of thyroid cancer.MethodsPlasma IgG N-glycome analysis was applied to a discovery cohort followed by independent validation. IgG N-glycan profiles were obtained using a robust quantitative strategy based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. IgG N-glycans were relatively quantified, and classification performance was evaluated based on directly detected and derived glycan traits.ResultsFour directly detected glycans were significantly changed in thyroid cancer patients compared to that in non-cancer controls. Derived glycan traits and a classification glycol-panel were generated based on the directly detected glycan traits. In the discovery cohort, derived trait BN (bisecting type neutral N-glycans) and the glyco-panel showed potential in distinguishing between thyroid cancer and non-cancer controls with AUCs of 0.920 and 0.917, respectively. The diagnostic potential was further validated. Derived trait BN and the glycol-panel displayed “accurate” performance (AUC>0.8) in discriminating thyroid cancer from benign thyroid nodules and healthy controls in the validation cohort. Meanwhile, derived trait BN and the glycol-panel also showed diagnostic potential in detecting early-stage thyroid cancer.ConclusionsIgG N-glycome analysis revealed N-glycomic differences that allow classification of thyroid cancer from non-cancer controls. Our results suggested that derived trait BN and the classification glyco-panel rather than single N-glycans may serve as candidate biomarkers for further validation.

Aberrantly glycosylated IgG elicits pathogenic signaling in podocytes and signifies lupus nephritis.

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of “liquid kidney biopsy” approaches to diagnose LN.

Extensive weight loss reduces glycan age by altering IgG N-glycosylation

BackgroundObesity, 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.MethodsIgG 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.ResultsLow-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).ConclusionsAltogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced glycan and biological age.

Glycosylation of recombinant rabbit immunoglobulins influences protease susceptibility as shown by comprehensive mass spectrometric glycan analysis.

Recombinant immunoglobulins (rIgGs) have become increasingly important as therapeutic agents and diagnostic tools in recent years. Genetic engineering allows the introduction of non-natural features such as the Sortase motif for site-directed labeling. In this study, the enzyme Sortase A (SrtA) was used for the proteolytic cleavage of rIgGs to produce their biotinylated Fab fragments by locating the cleavage site close to the hinge region. However, SrtA cleavage of engineered rabbit IgGs (rRb-IgGs) derived from human embryonic kidney (HEK) 293 cells showed significantly lower yields compared with their mouse counterparts. Nonrecombinant Rb-IgGs have N- and O-glycans, and the presence of O-glycans close to the hinge region of the rRb-IgGs might affect the susceptibility of these antibodies to SrtA cleavage. In addition, the glycosylation pattern of rIgGs differs depending on the host cell used for expression. Therefore, we analyzed the N- and O-glycans of various rRb-IgGs expressed in HEK293 cells, detecting and quantifying 13 different N-glycan and 3 different O-glycan structures. The distribution of the different detected glycoforms in our rRb-IgG N-glycan analysis is in agreement with previous studies on recombinant human IgG N-glycans, confirming the hypothesis that the host cell defines the glycosylation of the recombinant produced IgGs. O-glycosylation could be mapped onto the threonine residue within the hinge region sequence XPTCPPPX, as already described previously for nonrecombinant Rb-IgGs. Substitution of this threonine allowed an almost complete Fab fragment cleavage. Therefore, we could confirm the hypothesis that the O-glycans affect the SrtA activity, probably due to steric hindrance.

Glycosylation Alterations in Multiple Sclerosis Show Increased Proinflammatory Potential.

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.

Dynamic alterations in serum IgG N-glycan profiles in the development of colitis-associated colon Cancer in mouse model

BackgroundAlternative glycosylation of serum IgG has been shown to be closely associated with colorectal cancer (CRC). Currently, a dynamic study which can not only minimize the influence of genetic background, environment and other interfering factors during cancer development, but also focus on investigating carcinogenic characteristics of IgG glycan is lacking. MethodsSerum IgG N-glycans were characterized at four stages of CRC development by ultra-performance liquid chromatography in a typical colitis-related CRC mouse model induced by azoxymethane-dextran sodium sulfate. Furthermore, the expression of related glycosyltransferases in splenic B lymphocytes at the corresponding time was also assessed. ResultsThe relative abundance of seven IgG glycans, which can be classified as monoantennary, core fucose, sialic acid, galactose and bisecting, was changed during tumor growth. The abundance of some glycans was altered during the first stage of cancer induction. Correspondingly, the expression of glycosyltransferases in splenic B lymphocytes and different tissues in cancer groups was also decreased compared to that in controls. ConclusionsThis study represents the comprehensive analysis of IgG glycosylation in the dynamic process of colitis-associated CRC. To our knowledge, this is the first report that the expression of glycosyltransferases in mouse splenic B lymphocytes is consistent or inconsistent with the alterations of IgG N-glycans, and the variation tendency is tissue nonspecific. General SignificanceProviding a novel approach to identify the IgG glycans related to the development of CRC and laying a foundation for research on structure and function of glycans using mouse.

Quantitative analysis of serum-based IgG agalactosylation for tuberculosis auxiliary diagnosis.

Tuberculosis (TB) is the leading infectious cause of mortality worldwide, especially in developing countries. However, effective means for TB diagnosis, especially for bacillus-negative (Bn) TB laboratory diagnosis, are urgently needed. In the present study, serum IgG from each tuberculosis patients and healthy controls was purified using affinity chromatography. The samples were then analyzed using mass spectrometry (MS) and ultraperformance liquid chromatography (UPLC) methods. We quantitatively assessed the changes of serum IgG galactosylation in 567 human serum samples including 377 pulmonary TB patients and 190 healthy donors (HDs). We found significantly more agalactosylated (G0) vs monogalactosylated (G1) and digalactosylated (G2) N-glycans of IgG in TB patients, including smear-negative TB patients, than in HDs. The detection rate of TB diagnostic performance by MS for IgG-Gal ratio G0/(G1+G2×2) is 90.48% for bacillus-positive (Bp) and 73.16% for Bn TB patients. Further, combination of MS method with other routine laboratory TB diagnostic methods significantly increased the detection rate to 91.01%-98.39%. Similar results were observed in Mycobacterium tuberculosis (M. tb) infection mouse models. The decrease in galactosylation of IgG in TB patients was also qualitatively confirmed using specific lectin blot assay. Using the above techniques, we can discriminate the content of IgG G0 with terminal N-acetylglucosamine and IgG-Gal ratio G0/(G1+G2×2) between TB patients and HDs. Our data suggest that quantitative analysis of serum-based IgG-Gal ratio G0/(G1+G2×2) could be used for TB auxiliary diagnosis with high effectiveness and feasibility and its combination with other routine laboratory TB diagnostic methods could remarkably improve the detection rate.

Dysregulated Antibody, Natural Killer Cell and Immune Mediator Profiles in Autoimmune Thyroid Diseases.

The pathogenesis of autoimmune thyroid diseases (AITD) is poorly understood and the association between different immune features and the germline variants involved in AITD are yet unclear. We previously observed systemic depletion of IgG core fucosylation and antennary α1,2 fucosylation in peripheral blood mononuclear cells in AITD, correlated with anti-thyroid peroxidase antibody (TPOAb) levels. Fucose depletion is known to potentiate strong antibody-mediated NK cell activation and enhanced target antigen-expressing cell killing. In autoimmunity, this may translate to autoantibody-mediated immune cell recruitment and attack of self-antigen expressing normal tissues. Hence, we investigated the crosstalk between immune cell traits, secreted proteins, genetic variants and the glycosylation patterns of serum IgG, in a multi-omic and cross-sectional study of 622 individuals from the TwinsUK cohort, 172 of whom were diagnosed with AITD. We observed associations between two genetic variants (rs505922 and rs687621), AITD status, the secretion of Desmoglein-2 protein, and the profile of two IgG N-glycan traits in AITD, but further studies need to be performed to better understand their crosstalk in AITD. On the other side, enhanced afucosylated IgG was positively associated with activatory CD335- CD314+ CD158b+ NK cell subsets. Increased levels of the apoptosis and inflammation markers Caspase-2 and Interleukin-1α positively associated with AITD. Two genetic variants associated with AITD, rs1521 and rs3094228, were also associated with altered expression of the thyrocyte-expressed ligands known to recognize the NK cell immunoreceptors CD314 and CD158b. Our analyses reveal a combination of heightened Fc-active IgG antibodies, effector cells, cytokines and apoptotic signals in AITD, and AITD genetic variants associated with altered expression of thyrocyte-expressed ligands to NK cell immunoreceptors. Together, TPOAb responses, dysregulated immune features, germline variants associated with immunoactivity profiles, are consistent with a positive autoreactive antibody-dependent NK cell-mediated immune response likely drawn to the thyroid gland in AITD.

Antibody-mediated trapping in biological hydrogels is governed by sugar-sugar hydrogen bonds

N-glycans on IgG and IgM antibodies (Ab) facilitate Ab-mediated crosslinking of viruses and nanoparticles to the major structural elements of mucus and basement membranes. Nevertheless, the chemical moieties in these biological hydrogel matrices to which Ab can bind remain poorly understood. To gain insights into the chemistries that support Ab-matrix interactions, we systematically evaluated IgG- and IgM-mediated trapping of nanoparticles in different polysaccharide-based biogels with unique chemical features. In agarose, composed of alternating d-galactose and 3,6-anhydro-l-galactopyranose (i.e. hydroxyl groups only), anti-PEG IgM but not anti-PEG IgG trapped PEGylated nanoparticles. In alginate, comprised of homopolymeric blocks of mannuronate and guluronate (i.e. both hydroxyl and carboxyl groups), both IgG and IgM trapped PEGylated nanoparticles. In contrast, chitosan, comprised primarily of glucosamine (i.e. both hydroxyl and primary amine groups), did not facilitate either IgG- or IgM-mediated trapping. IgG-mediated trapping in alginate was abrogated upon removal of IgG N-glycans, whereas IgM-mediated trapping was eliminated in agarose but not alginate upon desialylation. These results led us to propose a model in which hydrogen bonding between carboxyl and hydroxyl groups of glycans on both Ab and matrix facilitates Ab-mediated trapping of pathogens in biogels. Our work here offers a blueprint for designing de novo hydrogels that could harness Ab-matrix interactions for various biomedical and biological applications. Statement of SignificanceHere, we interrogated the molecular mechanism of antibody-mediated trapping to address what are the chemical moieties on biogels that are essential for facilitating trapping in biogels. We systematically evaluated the potencies of IgG and IgM to trap nanoparticles in different polysaccharide-based biogels with unique and highly defined chemical moieties: hydroxyl groups (agarose), amine groups (chitosan), and carboxyl groups (alginate). We discovered that only hydroxyl/carboxyl hydrogen bonds (and stronger) are sufficiently strong enough to facilitate antibody-mediated trapping; weaker hydroxyl/hydroxyl bonds or hydroxyl/amine bonds fail to adequately slow particles. Our findings presents the first blueprint for how to engineer de novo biogels that are capable of harnessing antibodies to immobilize foreign entities in the biogels, for applications ranging from infectious disease to contraception to purification processes.

Prediction of neoadjuvant chemotherapeutic efficacy in patients with locally advanced gastric cancer by serum IgG glycomics profiling

BackgroundNeoadjuvant chemotherapy (NACT) could improve prognosis and survival quality of patients with local advanced gastric cancer (LAGC) by providing an opportunity of radical operation for them. However, no effective method could predict the efficacy of NACT before surgery to avoid the potential toxicity, time-consuming and economic burden of ineffective chemotherapy. Some research has been investigated about the correlation between serum IgG glycosylation and gastric cancer, but the question of whether IgG glycome can reflect the tumor response to NACT is still unanswered.MethodSerum IgG glycome profiles were analyzed by Ultra Performance Liquid Chromatography in a cohort comprised of 49 LAGC patients of which 25 were categorized as belonging to the NACT response group and 24 patients were assigned to the non-response group. A logistic regression model was constructed to predict the response rate incorporating clinical features and differential N-glycans, while the precision of model was assessed by receiver operating characteristic (ROC) analysis.ResultsIgG N-glycome analysis in pretreatment serum of LAGC patients comprises 24 directly detected glycans and 17 summarized traits. Compared with IgG glycans of non-response group, agalactosylated N-glycans increased while monosialylated N-glycans and digalactosylated N-glycans decreased in the response group. We constructed a model combining patients’ age, histology, chemotherapy regimen, GP4(H3N4F1), GP6(H3N5F1), and GP18(H5N4F1S1), and ROC analysis showed this model has an accurate prediction of NACT response (AUC = 0.840) with the sensitivity of 64.00% and the specificity of 100%.ConclusionWe here firstly present the profiling of IgG N-glycans in pretreatment serum of LAGC. The alterations in IgG N-glycome may be personalized biomarkers to predict the response to NACT in LAGC and help to illustrate the relationship between immunity and effect of NACT.

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.

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.

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography.

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Profiling of isomer-specific IgG N-glycosylation in cohort of Chinese colorectal cancer patients

BackgroudGiven the increasing morbidity and mortality of colorectal cancer (CRC), it is urgent to develop a noninvasive screening strategy for early diagnosis of CRC. Altered IgG glycosylation is associated with CRC progression, whereas the association of IgG isomeric glycosylation with CRC were not investigated. MethodsMethylamidation of IgG N-glycans was conducted prior to PGC-based nanoLC-ESI-MS/MS analysis. Data processing was operated by a self-developed application based on MATLAB solution. Statistical analysis including K–S test, t-test, ROC curve and OPLS-DA were successively performed. Additionally, an independent set was utilized to validate the results. ResultsTotal 28 IgG glycans and 79 compositional isomers were identified, over half of which are firstly identified so far. Statistical analysis showed that CRC associates with increase in IgG agalactosylation, decrease in IgG sialylation and fucosylation of sialylated glycans. Additionally, it was found that three compositional isomers (H3N4F1-a, H3N4F1-b and H4N3S1F1-e) could distinguish CRC and early stages from controls with an accurate area under the receiver operating characteristic curve. Significantly, these results were validated in an independent set by multivariate statistical analysis. ConclusionsThis is the first comprehensively profiling of isomer-specific IgG N-glycosylation, which could differentiate normal controls from colorectal disease patients. The candidate IgG glyco-biomarkers provide important screening indicators for early diagnosis of CRC. General significanceColorectal cancer progression is strongly associated with isomer-specific IgG N-glycosylation.

N-glycomic Profile in Combat Related Post-Traumatic Stress Disorder.

Post-traumatic stress disorder (PTSD) develops in a portion of individuals exposed to extreme trauma. Glycosylation is a post-translational modification that affects protein functions and is altered in various pathophysiological states and aging. There are still no validated biomarkers of PTSD. The aim of this study was to evaluate the N-glycomic profile in 543 male Caucasian individuals (299 veterans with PTSD and 244 control subjects). The study included discovery (N = 233) and replication (N = 310) cohort. Hydrophilic interaction HPLC and ultra-performance liquid chromatography were used to separate and detect 39 plasma and 24 IgG N-glycan species, respectively. All results were corrected for the effects of age and multiple testing. Significant results included only significantly altered N-glycans in cases/controls in both cohorts, in the same direction. Results showed that six plasma N-glycans (four increased and two decreased) were altered in PTSD vs. controls in both cohorts, but IgG N-glycans were similar between groups. The severity of PTSD was not associated with different plasma N-glycans. This is the first study detecting alterations in plasma N-glycans in PTSD. These N-glycans are also associated with other neuropsychiatric disorders and inflammation, suggesting possible shared glycosylation mechanisms.

A streamlined workflow for twoplexing of N-linked glycan analysis using light (12C6) and heavy (13C6) isotopologues of 3-aminobenzenesulfonic acid

Comparative glycosylation analysis of biopharmaceuticals requires the development of methods that deliver the necessary throughput, support structural elucidation and relative quantitation of glycans released from therapeutics. The current study presents the development and applicability assessment of a twoplex approach using light and heavy isotopolouges of 3-aminobenzenesulfonic acid (3-ASA) under wet labeling conditions followed by UHPLC-MS analysis in data dependent acquisition mode. Excellent labelling efficiency, >90%, was achieved for both the light and heavy variants of the reagent. Glycan distributions of two human IgG lots labeled by light and heavy isotopolouges were identical, demonstrating no labeling bias introduced by either of the isotopologues. Peak area distributions of glycan profiles of two human IgG lots were compared to 2-aminobenzamide (2-AB) and RapiFluor-MS protocols. The comparison led to identical results in peak area distribution across the three dyes, but differences in chromatographic selectivity attributed to the different tags. MS1 based relative quantitation was further validated by releasing glycans from the same lot of human IgG, with glycan pools obtained labeled with light and heavy isotopologues separately, followed by mixing and clean-up of the same amount of light and heavy labeled glycan pools. MS analyses of each glycan resulted in a ratio of light and heavy XIC in the range of 0.97 ≤ x ≤ 1.05, demonstrating the method is amenable for the relative quantitation of glycans. Excellent correlation between the relative quantitation data of N-glycans from two human IgG N-glycan pools using the twoplex approach and ratios from peak area distribution calculated from the fluorescent chromatogram was observed (r = 0.986), further corroborating the reliability of the method and its potential applicability in the biopharmaceutical industry. Highly informative HCD-MS2 spectra dominated mostly by Y- and Z-type single and double glycosidic fragment ions facilitate structural interpretation of the oligosaccharides.