1 Scientific RepoRts | 6:24363 | DOI: 10.1038/srep24363 www.nature.com/scientificreports DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins Marija Klasić 1 , Jasminka Krištić 2 , Petra Korać 1 , tomislav Horvat 1 , Dora Markulin 1 , Aleksandar Vojta 1 , Karli R. Reiding 3 , Manfred Wuhrer 3,4 , Gordan Lauc 2,5 & Vlatka Zoldoš 1 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 diferent concentrations of 5-aza- 2′-deoxycytidine (5-aza-2dC) diferentially afected global genome methylation and induced diferent 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 proile 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. Glycosylation is an important post-translational modiication that can signiicantly contribute to variability in protein structures 1,2 . While many protein modiications, such as phosphorylation, generally function as on/of switches, glycosylation is more diverse. A number of diferent glycans can be attached to the same glycosylation site, resulting in alteration of both glycoprotein structure and function. herefore, glycosylation is a vital func- tional and regulatory modiication of proteins and almost all membrane and secreted proteins are covalently modiied by complex oligosaccharides. Glycans attached to proteins or lipids deine the large part of the surface of mammalian cells, making glycans the major determinants in cellular interaction and communication 3 . Moreover, glycans integrate genetic and environmental factors, and are thus closely associated with complex diseases 4 . Glycans are synthesized in a complex biochemical chain of reactions, involving many enzymes and other pro- teins 5 . Classical glyco-genes (coding for glycosidases, glycosyltransferases, etc.) represent only a part of this large metabolic pathway that also includes numerous transcription factors, transport proteins, ion channels, Golgi organizers, etc. Current studies indicate that glycosylation pathways include at least three to four times more genes than the currently listed 800 glyco-genes, suggesting that more than 10% of the genome may be involved in glycosylation 6–8 . Regulation of glycosylation seems to be very complex and still not fully understood–it is under the inluence of genetic and epigenetic factors as well as various internal and external environmental fac- tors 2,4,5,9–11 . Regulatory mechanisms may induce changes in the activity, relative abundance and/or localization of any of the enzymes involved in glycan biosynthesis afecting the inal structure of a glycan. Nevertheless, little 1 University of Zagreb faculty of Science, Zagreb, croatia. 2 Genos Glycoscience Research Laboratory, Zagreb, croatia. 3 center for Proteomics and Metabolomics, Leiden University Medical center, Leiden, the netherlands. 4 Division of BioAnalytical chemistry, VU University Amsterdam, Amsterdam, the netherlands. 5 University of Zagreb faculty of Pharmacy and Biochemistry, Zagreb, croatia. correspondence and requests for materials should be addressed to V.Z. (email: vzoldos@biol.pmf.hr) Received: 10 September 2015 Accepted: 23 March 2016 Published: 13 April 2016 opeN