High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

Baseline IgG-Fc N-glycosylation profile is associated with long-term outcome in a cohort of early inflammatory arthritis patients.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease for which prediction of long-term prognosis from disease's outset is not clinically feasible. The importance of immunoglobulin G (IgG) and its Fc N-glycosylation in inflammation is well-known and studies described its relevance for several autoimmune diseases, including RA. Herein we assessed the association between IgG N-glycoforms and disease prognosis at 2 years in an early inflammatory arthritis cohort. METHODS: Sera from 118 patients with early inflammatory arthritis naïve to treatment sampled at baseline were used to obtain IgG Fc glycopeptides, which were then analyzed in a subclass-specific manner by liquid chromatography coupled to mass spectrometry (LC-MS). Patients were prospectively followed and a favorable prognosis at 2 years was assessed by a combined index as remission or low disease activity (DAS28 < 3.2) and normal functionality (HAQ ≤ 0.25) while on treatment with conventional synthetic DMARDs and never used biologic DMARDs. RESULTS: We observed a significant association between high levels of IgG2/3 Fc galactosylation (effect 0.627 and adjusted p value 0.036 for the fully galactosylated glycoform H5N4F1; effect -0.551 and adjusted p value 0.04963 for the agalactosylated H3N4F1) and favorable outcome after 2 years of treatment. The inclusion of IgG glycoprofiling in a multivariate analysis to predict the outcome (with HAQ, DAS28, RF, and ACPA included in the model) did not improve the prognostic performance of the model. CONCLUSION: Pending confirmation of these findings in larger cohorts, IgG glycosylation levels could be used as a prognostic marker in early arthritis, to overcome the limitations of the current prognostic tools.

Distinct Longitudinal Changes in Immunoglobulin G N-Glycosylation Associate with Therapy Response in Chronic Inflammatory Diseases.

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.

Immunoglobulin G glycans - Biomarkers and molecular effectors of aging.

Immunoglobulin G (IgG) antibodies are post-translationally modified by the addition of complex carbohydrate molecules - glycans, which have profound effects on the IgG function, most significantly as modulators of its inflammatory capacity. Therefore, it is not surprising that the changes in IgG glycosylation pattern are associated with various physiological states and diseases, including aging and age-related diseases. Importantly, within the inflammaging concept, IgG glycans are considered not only biomarkers but one of the molecular effectors of the aging process. The exact mechanism by which they exert their function, however, remains unknown. In this review, we list and comment on, to our knowledge, all studies that examined changes in IgG glycosylation during aging in humans. We focus on the information obtained from studies on general population, but we also cover the insights obtained from studies of long-lived individuals and people with age-related diseases. We summarize the current knowledge on how levels of different IgG glycans change with age (i.e., the extent and direction of the change with age) and discuss the potential mechanisms and possible functional roles of changes in IgG glycopattern that accompany aging.

Association Between Human Gut Microbiome and N-Glycan Composition of Total Plasma Proteome.

Being one of the most dynamic entities in the human body, glycosylation of proteins fine-tunes the activity of the organismal machinery, including the immune system, and mediates the interaction with the human microbial consortium, typically represented by the gut microbiome. Using data from 194 healthy individuals, we conducted an associational study to uncover potential relations between the gut microbiome and the blood plasma N-glycome, including N-glycome of immunoglobulin G. While lacking strong linkages on the multivariate level, we were able to identify associations between alpha and beta microbiome diversity and the blood plasma N-glycome profile. Moreover, for two bacterial genera, namely, Bilophila and Clostridium innocuum, significant associations with specific glycans were also shown. The study's results suggest a non-trivial, possibly weak link between the total plasma N-glycome and the gut microbiome, predominantly involving glycans related to the immune system proteins, including immunoglobulin G. Further studies of glycans linked to microbiome-related proteins in well-selected patient groups are required to conclusively establish specific associations.

Immunoglobulin G Glycosylation in Diseases.

Changes in immunoglobulin G (IgG) glycosylation pattern have been observed in a vast array of auto- and alloimmune, infectious, cardiometabolic, malignant, and other diseases. This chapter contains an updated catalog of over 140 studies within which IgG glycosylation analysis was performed in a disease setting. Since the composition of IgG glycans is known to modulate its effector functions, it is suggested that a changed IgG glycosylation pattern in patients might be involved in disease development and progression, representing a predisposition and/or a functional effector in disease pathology. In contrast to the glycopattern of bulk serum IgG, which likely relates to the systemic inflammatory background, the glycosylation profile of antigen-specific IgG probably plays a direct role in disease pathology in several infectious and allo- and autoimmune antibody-dependent diseases. Depending on the specifics of any given disease, IgG glycosylation read-out might therefore in the future be developed into a useful clinical biomarker or a supplementary to currently used biomarkers.

Glycosylation and Aging.

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

Targeting B cells in the pre-phase of systemic autoimmunity globally interferes with autoimmune pathology.

Systemic lupus erythematosus (SLE) is characterized by a loss of self-tolerance, systemic inflammation, and multi-organ damage. While a variety of therapeutic interventions are available, it has become clear that an early diagnosis and treatment may be key to achieve long lasting therapeutic responses and to limit irreversible organ damage. Loss of humoral tolerance including the appearance of self-reactive antibodies can be detected years before the actual onset of the clinical autoimmune disease, representing a potential early point of intervention. Not much is known, however, about how and to what extent this pre-phase of disease impacts the onset and development of subsequent autoimmunity. By targeting the B cell compartment in the pre-disease phase of a spontaneous mouse model of SLE we now show, that resetting the humoral immune system during the clinically unapparent phase of the disease globally alters immune homeostasis delaying the downstream development of systemic autoimmunity.

Effects of Estradiol on Immunoglobulin G Glycosylation: Mapping of the Downstream Signaling Mechanism.

Glycans attached to immunoglobulin G (IgG) directly affect this antibody effector functions and regulate inflammation at several levels. The composition of IgG glycome changes significantly with age. In women, the most notable change coincides with the perimenopausal period. Aiming to investigate the effect of estrogen on IgG glycosylation, we analysed IgG and total serum glycomes in 36 healthy premenopausal women enrolled in a randomized controlled trial of the gonadotropin-releasing hormone analogue (GnRHAG) leuprolide acetate to lower gonadal steroids to postmenopausal levels and then randomized to transdermal placebo or estradiol (E2) patch. The suppression of gonadal hormones induced significant changes in the IgG glycome, while E2 supplementation was sufficient to prevent changes. The observed glycan changes suggest that depletion of E2 primarily affects B cell glycosylation, while liver glycosylation stays mostly unchanged. To determine whether previously identified IgG GWAS hits RUNX1, RUNX3, SPINK4, and ELL2 are involved in downstream signaling mechanisms, linking E2 with IgG glycosylation, we used the FreeStyle 293-F transient system expressing IgG antibodies with stably integrated CRISPR/dCas9 expression cassettes for gene up- and downregulation. RUNX3 and SPINK4 upregulation using dCas9-VPR resulted in a decreased IgG galactosylation and, in the case of RUNX3, a concomitant increase in IgG agalactosylation.

N-glycosylation analysis of mouse immunoglobulin G isolated from dried blood spots.

The association of immunoglobulin G (IgG) glycosylation changes with various human diseases and physiological conditions is well established. Since the mechanistical explanation of the regulation of IgG glycosylation and its functional role in these various states is still missing, the eyes of the biomedical community are now turned towards animal models, which enable intervention studies necessary for conclusions on causality. Mice are recognized and used as a good experimental model for human IgG glycosylation. However, smaller blood volumes, low IgG concentrations at young ages (which are most often used in mice experiments) and multiple sampling protocols during the course of longitudinal studies would profit from a robust workflow for mouse IgG glycome analysis from minute amounts of starting material, collected through a simple sampling procedure. For this purpose, we have developed a protocol for analysis of total N-glycans of IgG isolated from mouse dried blood spots (DBS), which we report here. We show that mouse DBS are a good source of material for IgG N-glycan analysis by multiplexed capillary gel electrophoresis with laser-induced fluorescence (xCGE-LIF).

Effects of estradiol on biological age measured using the glycan age index.

Glycan age is a recently developed biomarker based on glycans attached to immunoglobulin G (IgG). In large population cohorts, glycan age associates well with lifestyle and disease-risk biomarkers, while some studies suggested that glycan changes precede development of several age-associated diseases. In this study we evaluated effects of estrogen on the glycan age. Gonadal hormones were suppressed in 36 healthy young women by gonadotropin releasing hormone agonist therapy for 6 months. In 15 of them estradiol was supplemented, while 21 received placebo resulting in very low estrogen levels during intervention. IgG was isolated from plasma samples before intervention, after 6 months of intervention and after subsequent 4-month recovery. Deprivation of gonadal hormones resulted in median increase of glycan age for 9.1 years (IQR 6.8 - 11.5 years, p = 3.73×10-8), which was completely prevented by transdermal estradiol therapy (change in glycan age = -0.23 years, IQR (-2.20 - 2.98). After the recovery period glycan age returned to baseline values in both groups. These results suggest that IgG glycans and consequently also the glycan age are under strong influence of gonadal hormones and that estradiol therapy can prevent the increase of glycan age that occurs in the perimenopausal period.

Fc-Linked IgG N-Glycosylation in FcγR Knock-Out Mice.

Immunoglobulin G (IgG) is the most abundant immunoglobulin isotype in the blood and is involved in the pathogenesis and progression of various diseases. Glycosylation of the IgG fragment crystallizable (Fc) region is shown to vary in different physiological and pathological states. Fc N-glycan composition can alter the effector functions of IgG by modulating its affinity for ligands, such as Fcγ receptors (FcγRs). However, it is not known whether IgG glycosylation is affected by the available repertoire of FcγRs, and if the Fc-linked N-glycome can compensate for modulation of the IgG-FcγR interaction. To explore this, we examined the subclass-specific Fc IgG glycoprofiles of healthy male and female FcγR knock-out mice on C57BL/6 and BALB/c backgrounds. We observed slight changes in IgG Fc N-glycan profiles in different knock-outs; however, it seems that the strain background and sex have a stronger effect on N-glycosylation of IgG Fc regions than the FcγR repertoire.

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.

Decreased Immunoglobulin G Core Fucosylation, A Player in Antibody-dependent Cell-mediated Cytotoxicity, is Associated with Autoimmune Thyroid Diseases.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

Comprehensive N-glycosylation analysis of immunoglobulin G from dried blood spots.

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.

Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo.

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo.

MIgGGly (mouse IgG glycosylation analysis) - a high-throughput method for studying Fc-linked IgG N-glycosylation in mice with nanoUPLC-ESI-MS.

Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.

Immunoglobulin G glycosylation in aging and diseases.

The Immunoglobulin G (IgG) glycome is well known for its heterogeneity and shows a significant degree of variation within populations. IgG glycome composition is influenced both by genes and by environment, making it an excellent biomarker of a person's general health state, i.e. biological age. IgG glycosylation appears to be highly regulated, both during homeostasis and in cases of its disturbance. Changes in IgG glycosylation patterns have been observed in aging and in various diseases. Differential IgG glycosylation is known to modulate IgG effector functions and is involved in disease development and progression, representing both a predisposition and a functional mechanism involved in disease pathology. This makes IgG glycosylation analysis a promising add-on to improve existing disease biomarkers.