Dora Markulin

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.

Promoter methylation of the MGAT3 and BACH2 genes correlates with the composition of the immunoglobulin G glycome in inflammatory bowel disease

BackgroundMany genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. MethodsUsing bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively.ResultsWe found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn’s disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19+ B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly.ConclusionsOur results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3+ T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.