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.

Plasma N-glycome composition associates with chronic low back pain.

BACKGROUND: Low back pain (LBP) is the symptom of a group of syndromes with heterogeneous underlying mechanisms and molecular pathologies, making treatment selection and patient prognosis very challenging. Moreover, symptoms and prognosis of LBP are influenced by age, gender, occupation, habits, and psychological factors. LBP may be characterized by an underlying inflammatory process. Previous studies indicated a connection between inflammatory response and total plasma N-glycosylation. We wanted to identify potential changes in total plasma N-glycosylation pattern connected with chronic low back pain (CLBP), which could give an insight into the pathogenic mechanisms of the disease. METHODS: Plasma samples of 1128 CLBP patients and 760 healthy controls were collected in clinical centers in Italy, Belgium and Croatia and used for N-glycosylation profiling by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) after N-glycans release, fluorescent labeling and clean-up. Observed N-glycosylation profiles have been compared with a cohort of 126 patients with acute inflammation that underwent abdominal surgery. RESULTS: We have found a statistically significant increase in the relative amount of high-branched (tri-antennary and tetra-antennary) N-glycan structures on CLBP patients' plasma glycoproteins compared to healthy controls. Furthermore, relative amounts of disialylated and trisialylated glycan structures were increased, while high-mannose and glycans containing bisecting N-acetylglucosamine decreased in CLBP. CONCLUSIONS: Observed changes in CLBP on the plasma N-glycome level are consistent with N-glycosylation changes usually seen in chronic inflammation. GENERAL SIGNIFICANCE: To our knowledge, this is a first large clinical study on CLBP patients and plasma N-glycome providing a new glycomics perspective on potential disease pathology.

Profiling and genetic control of the murine immunoglobulin G glycome.

Immunoglobulin G (IgG) glycosylation is essential for function of the immune system, but the genetic and environmental factors that underlie its inter-individual variability are not well defined. The Collaborative Cross (CC) genetic resource harnesses over 90% of the common genetic variation of the mouse. By analyzing the IgG glycome composition of 95 CC strains, we made several important observations: (i) glycome variation between mouse strains was higher than between individual humans, despite all mice having the same environmental influences; (ii) five genetic loci were found to be associated with murine IgG glycosylation; (iii) variants outside traditional glycosylation site motifs affected glycome variation; (iv) bisecting N-acetylglucosamine (GlcNAc) was produced by several strains although most previous studies have reported the absence of glycans containing the bisecting GlcNAc on murine IgGs; and (v) common laboratory mouse strains are not optimal animal models for studying effects of glycosylation on IgG function.

IgG glycosylation and DNA methylation are interconnected with smoking.

BACKGROUND: Glycosylation is one of the most common post-translation modifications with large influences on protein structure and function. The effector function of immunoglobulin G (IgG) alters between pro- and anti-inflammatory, based on its glycosylation. IgG glycan synthesis is highly complex and dynamic. METHODS: With the use of two different analytical methods for assessing IgG glycosylation, we aim to elucidate the link between DNA methylation and glycosylation of IgG by means of epigenome-wide association studies. In total, 3000 individuals from 4 cohorts were analyzed. RESULTS: The overlap of the results from the two glycan measurement panels yielded DNA methylation of 7 CpG-sites on 5 genomic locations to be associated with IgG glycosylation: cg25189904 (chr.1, GNG12); cg05951221, cg21566642 and cg01940273 (chr.2, ALPPL2); cg05575921 (chr.5, AHRR); cg06126421 (6p21.33); and cg03636183 (chr.19, F2RL3). Mediation analyses with respect to smoking revealed that the effect of smoking on IgG glycosylation may be at least partially mediated via DNA methylation levels at these 7 CpG-sites. CONCLUSION: Our results suggest the presence of an indirect link between DNA methylation and IgG glycosylation that may in part capture environmental exposures. GENERAL SIGNIFICANCE: An epigenome-wide analysis conducted in four population-based cohorts revealed an association between DNA methylation and IgG glycosylation patterns. Presumably, DNA methylation mediates the effect of smoking on IgG glycosylation.

DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins.

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.

Glycans - the third revolution in evolution.

The development and maintenance of a complex organism composed of trillions of cells is an extremely complex task. At the molecular level every process requires a specific molecular structures to perform it, thus it is difficult to imagine how less than tenfold increase in the number of genes between simple bacteria and higher eukaryotes enabled this quantum leap in complexity. In this perspective article we present the hypothesis that the invention of glycans was the third revolution in evolution (the appearance of nucleic acids and proteins being the first two), which enabled the creation of novel molecular entities that do not require a direct genetic template. Contrary to proteins and nucleic acids, which are made from a direct DNA template, glycans are product of a complex biosynthetic pathway affected by hundreds of genetic and environmental factors. Therefore glycans enable adaptive response to environmental changes and, unlike other epiproteomic modifications, which act as off/on switches, glycosylation significantly contributes to protein structure and enables novel functions. The importance of glycosylation is evident from the fact that nearly all proteins invented after the appearance of multicellular life are composed of both polypeptide and glycan parts.

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27 × 10(-9)) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.