Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status

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. Nglycans 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 ␣ 2-6 sialylated N-glycans and "N,N-diacetyllactosamine" 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. Molecular & Cellular

N-glycan MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissue Enables the Delineation of Ovarian Cancer Tissues

“…Saladova et al, reported specific N-glycosylation changes on serum glycoproteins from ovarian cancer patients, including a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLe(x) on haptoglobin beta-chain, alpha1-acid glycoprotein and alpha1-antichymotrypsin (30). We have also observed membrane glycosylation changes between non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR3) (31). The "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of ␣ 2-6 sialylated N-glycans and "N,NЈ-diacetyl-lactosamine" type N-glycans were predominantly observed in serous ovarian cancer cell lines while absent in the noncancerous ovarian cells.…”

“…The agalactosylated bisecting structure of m/z 1339.4 was observed only in the necrotic tissue of the sample P1. 2-6 sialic acids were differentiated based on the chromatographic behavior and retention times of previous studies (31,38,39). Detailed structural characterization of the N-glycans was carried out by tandem MS fragmentation analysis.…”

Multi-level regulation of cellular glycosylation: from genes to transcript to enzyme to structure

“…cellular mRNA) levels are often used as a key indicator of what glycogenes are being translated and the levels of biosynthetic enzymes and glycans that result, the relationship is not straightforward. A number of investigators have attempted to relate transcript levels to glycoenzyme activity and carbohydrate structures, including the response of the system to perturbations [11,24–26]. Such studies have been conducted using human promyelocytic leukocytes differentiated to primary neutrophils [24], a limited panel of mouse tissue [11], mouse embryonic stem cells differentiated to embryoid bodies and endodermal cells [25], and epithelial ovarian cancer cell types [26].…”

“…A number of investigators have attempted to relate transcript levels to glycoenzyme activity and carbohydrate structures, including the response of the system to perturbations [11,24–26]. Such studies have been conducted using human promyelocytic leukocytes differentiated to primary neutrophils [24], a limited panel of mouse tissue [11], mouse embryonic stem cells differentiated to embryoid bodies and endodermal cells [25], and epithelial ovarian cancer cell types [26]. The broad conclusion of these studies is that glycogene, and more specifically glycosyltransferase, expression measurements in a number of cases can semi-quantitatively predict corresponding enzyme activity and cell-surface glycan expression.…”

Integrated glycomic analysis of ovarian cancer side population cells

“…In our MS analysis, glycans with terminal Glcs (glucose), which are usually related to glycoprotein quality control [25, 26] and exhibit higher expression in cancer [27, 28], were expressed more abundantly in SP cells. Increased hybrid N-glycans were detected in MP cells, indicating that the immediate products of GlcNAc transferase I were not further modified by other GlcNAc transferases.…”