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Zagreb, Croatia

Horvat T.,University of Zagreb | Muzinic A.,Genos Ltd | Barisic D.,University of Zagreb | Bosnar M.H.,Ruder Boskovic Institute | Zoldos V.,University of Zagreb
Biochimica et Biophysica Acta - General Subjects | Year: 2012

Background: Epigenetic changes play a role in all major events during tumorigenesis and changes in glycan structures are hallmarks of virtually every cancer. Also, proper N-glycosylation of membrane receptors is important in cell to cell and cell-environment communication. To study how modulation of epigenetic information can affect N-glycan expression we analyzed effects of epigenetic inhibitors on HeLa cell membrane N-glycome. Methods: HeLa cells were treated with DNA methylation (zebularin and 5-aza-2-deoxycytidine) and histone deacetylation (trichostatin A and Na-butyrate) inhibitors. The effects on HeLa cell membrane N-glycome were analyzed by hydrophilic interaction high performance liquid chromatography (HILIC). Results: Each of the four epigenetic inhibitors induced changes in the expression of HeLa cell membrane N-glycans that were seen either as an increase or a decrease of individual glycans in the total N-glycome. Compared to DNA methylation inhibitors, histone deacetylation inhibitors showed more moderate changes, probably due to their higher gene target selectivity. Conclusions: The results clearly show that composition of HeLa cell membrane N-glycome can be specifically altered by epigenetic inhibitors. General significance: Glycans on the cell membrane are essential elements of tumor cell's metastatic potential and are also an entry point for nearly all pathogenic microorganisms. Since epigenetic inhibitors used in this work are registered drugs, our results provide a new line of research in the application of these drugs as anticancer and antimicrobial agents. This article is part of a Special Issue entitled Glycoproteomics. © 2011 Elsevier B.V. All rights reserved. Source

ZoldoS V.,University of Zagreb | Horvat T.,University of Zagreb | Lauc G.,University of Zagreb | Lauc G.,Genos Ltd
Current Opinion in Chemical Biology | Year: 2013

Majority of eukaryotic proteins are glycosylated and their glycan moieties have numerous important structural, functional and regulatory roles. Because of structural complexity of glycans and technological limitations glycomics, and particularly glycoproteomics was not able to follow rapid progress in genomics and proteomics over last 30 years. However, the field of glycan has been progressing rapidly and first large-scale studies of the glycome have been completed recently. These studies have revealed significant differences in glycome composition between individuals, which may contribute to the human phenotypic variability. The current state-of-the-art in high-throughput glycomics and its integration with genomics, epigenomics and lipidomics is reviewed in this article. © 2012 Elsevier Ltd. Source

Lauc G.,Genos Ltd | Lauc G.,University of Zagreb | Zoldo V.,University of Zagreb
Molecular BioSystems | Year: 2010

The majority of molecular processes in higher organisms are performed by various proteins and are thus determined by genes that encode these proteins. However, a significant structural component of at least half of all cellular proteins is not a polypeptide encoded by a single gene, but an oligosaccharide (glycan) synthesized by a network of proteins, resulting from the expression of hundreds of different genes. Relationships between hundreds of individual proteins that participate in glycan biosynthesis are very complex which enables the influence of environmental factors on the final structure of glycans, either by direct effects on individual enzymatic processes, or by induction of epigenetic changes that modify gene expression patterns. Until recently, the complexity of glycan structures prevented large scale studies of protein glycosylation, but recent advances in both glycan analysis and genotyping technologies, enabled the first insights into the intricate field of complex genetics of protein glycosylation. Mutations which inactivate genes involved in the synthesis of common N-glycan precursors are embryonically lethal. However, mutations in genes involved in modifications of glycan antennas are common and apparently contribute largely to individual phenotypic variations that exist in humans and other higher organisms. Some of these variations can be recognized as specific glyco-phenotypes that might represent specific evolutionary advantages or disadvantages. They are however, amenable to environmental influences and are thus less pre-determined than classical Mendelian mutations. © 2010 The Royal Society of Chemistry. Source

Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders. Source

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs. Source

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