Presence of bisecting glcnac-modified proteins on ovarian cancer cells associates with elevated expression of the epigenetically regulated MGAT3 gene

Abstract

Aims: Changes in the glycosylation of membrane proteins are a common feature of malignant transformation including ovarian cancer development. However, the nature of these changes and their underlying molecular mechanisms are poorly understood. Methods: Membrane protein glycan extraction and LC-ESI-MS (liquid chromatography negative-ion electrospray ionization mass spectrometry for glycan characterization), qRT-PCR (gene expression profiling in cell lines), bisulfite sequencing (analysis of DNA methylation) and Western blotting (protein determination). Results: We identified characteristic glycan features that were unique to membrane proteins of ovarian cancer cells such as the exclusive presence of "bisecting N-acetyl-glucosamine (GlcNAc)" type Nglycans. The bisecting GlcNAc on N-glycosylated proteins is the product of MGAT3 (beta-1,4-N-acetylglucosaminyltransferase 3). The expression of both the MGAT3 gene and the corresponding enzyme was elevated in ovarian cancer cell lines (SKOV3, IGROV1, A2780, OVCAR3) as compared to normal ovarian surface epithelial (HOSE) cells. We found that elevated MGAT3 expression in ovarian cancer cells correlated with DNA hypomethylation at the transcription start site (TSS) of the MGAT3 promoter, suggesting an epigenetic regulatory mechanism for MGAT3. This finding was further confirmed by treating HOSE cells with DNA-methyltransferase inhibitor, 5-Aza, which resulted in increased MGAT3 expression, mirrored by a reduction of DNA methylation at the TSS. Conclusion: Elevated MGAT3 expression in ovarian cancer cells is epigenetically regulated by DNA methylation. The presence of specific N-glycan substructures in combination with the epigenetic regulation of their associated enzymes may stimulate the development of novel anti-glycan drug targets and clinical diagnostic tools in ovarian cancer.2 page(s