Abstract
Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of α 2-6 sialylated N-glycans and "N,N'-diacetyl-lactosamine" type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique "bisecting GlcNAc" type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic programming of their associated synthetic enzymes in ovarian cancer could potentially be used for the development of novel anti-glycan drug targets and clinical diagnostic tools.
Highlights
From the ‡Department of Chemistry & Biomolecular Sciences, Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, NSW 2109, Sydney, Australia; §Gynaecological Research Group, Department of Biomedicine, Women’s University Hospital Basel, University of Basel, Basel 4003, Switzerland; ¶Ovarian Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, NSW 2052, Sydney, Australia
To identify specific membrane N-glycan changes in serous ovarian cancer cell lines (SKOV 3, IGROV 1, A2780, and OVCAR 3) and non-cancerous ovarian surface epithelial cell lines (HOSE 6.3 and HOSE 17.1), global glycosylation profiles of the glycans released from total membrane proteins by PNGase F were acquired using mass spectrometry
We identify specific N-glycan alterations on the cell surface membrane proteins of serous ovarian cancer cell lines that correlate with differential gene expression of the corresponding glycosyltransferase-encoded genes
Summary
Materials—N-Glycosidase F (PNGase F, recombinant clone derived from Flavobacterium meningosepticum and expressed in Escherichia coli) and protease inhibitor mixture tablets were purchased from Roche Diagnostics (Basel, Switzerland). ␣ 2–3 sialidase enzyme (Glyko® Sialidase S, recombinant derived from Streptococcus pneumonia and expressed in Escherichia coli) was purchased from Prozyme (Hayward, CA). All cultures were free of mycoplasma, as determined by qualitative PCR using VenorGeM® Mycoplasma Detection Kit. Cell Membrane Preparation and Triton X-114 Phase Partitioning of Membrane Proteins—Approximately 4 ϫ 107 cells were washed twice with PBS and pelleted through centrifugation at 2500 ϫ g for 20 mins to remove excess culture media. Cell pellets were re-suspended with 2 ml of lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and protease inhibitor at pH 7.4) and stored on ice for 20 mins. A volume of 450 l of Tris binding buffer containing 1% (v/v) Triton X-114 was added to the suspended mixture, homogenized by pipetting and chilled on ice for 10 mins. Membrane proteins and glycoprotein standard (10 g of fetuin) were
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