NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submit- Avenue, Silver Spring, Maryland 20993; 22Glycoscience Research Laboratory, Genos, Borongajska cesta 83h, 10 000 Zagreb, Croatia; 23Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇ ic´ a 1, 10 000 Zagreb, Croatia; 24Department of Chemistry, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303; 25glyXera GmbH, Brenneckestrasse 20 * ZENIT / 39120 Magdeburg, Germany; 26Health Products and Foods Branch, Health Canada, AL 2201E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9 Canada; 27Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama Higashi-Hiroshima 739–8530 Japan; 28ImmunoGen, 830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College, ul. Michalowskiego 12, 31–126 Krakow, Poland; 30Department of Pathology, Johns Hopkins University, 400 N. Broadway Street Baltimore, Maryland 21287; 31Mass Spec Core Facility, KBI Biopharma, 1101 Hamlin Road Durham, North Carolina 27704; 32Division of Mass Spectrometry, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongwon-gu, Cheongju Chungbuk, 363–883 Korea (South); 33Advanced Therapy Products Research Division, Korea National Institute of Food and Drug Safety, 187 Osongsaengmyeong 2-ro Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 363–700, Korea (South); 34Center for Proteomics and Metabolomics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 35Ludger Limited, Culham Science Centre, Abingdon, Oxfordshire, OX14 3EB, United Kingdom; 36Biomolecular Discovery and Design Research Centre and ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, North Ryde, Australia; 37Proteomics, Central European Institute for Technology, Masaryk University, Kamenice 5, A26, 625 00 BRNO, Czech Republic; 38Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; 39Department of Biomolecular Sciences, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany; 40AstraZeneca, Granta Park, Cambridgeshire, CB21 6GH United Kingdom; 41Merck, 2015 Galloping Hill Rd, Kenilworth, New Jersey 07033; 42Analytical R&D, MilliporeSigma, 2909 Laclede Ave. St. Louis, Missouri 63103; 43MS Bioworks, LLC, 3950 Varsity Drive Ann Arbor, Michigan 48108; 44MSD, Molenstraat 110, 5342 CC Oss, The Netherlands; 45Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5–1 Higashiyama, Myodaiji, Okazaki 444–8787 Japan; 46Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuhoku, Nagoya 467–8603 Japan; 47Medical & Biological Laboratories Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464–0858 Japan; 48National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG United Kingdom; 49Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158–8501 Japan; 50New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938; 51New York University, 100 Washington Square East New York City, New York 10003; 52Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom; 53GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; 54Department of Chemistry, North Carolina State University, 2620 Yarborough Drive Raleigh, North Carolina 27695; 55Pantheon, 201 College Road East Princeton, New Jersey 08540; 56Pfizer Inc., 1 Burtt Road Andover, Massachusetts 01810; 57Proteodynamics, ZI La Varenne 20–22 rue Henri et Gilberte Goudier 63200 RIOM, France; 58ProZyme, Inc., 3832 Bay Center Place Hayward, California 94545; 59Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho Nakagyo-ku, Kyoto, 604 8511 Japan; 60Children’s GMP LLC, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1–5 Muromati 1-Chome, Nishiku, Kobe, 651–2241 Japan; 62Synthon Biopharmaceuticals, Microweg 22 P.O. Box 7071, 6503 GN Nijmegen, The Netherlands; 63Takeda Pharmaceuticals International Co., 40 Landsdowne Street Cambridge, Massachusetts 02139; 64Department of Chemistry and Biochemistry, Texas Tech University, 2500 Broadway, Lubbock, Texas 79409; 65Thermo Fisher Scientific, 1214 Oakmead Parkway Sunnyvale, California 94085; 66United States Pharmacopeia India Pvt. Ltd. IKP Knowledge Park, Genome Valley, Shamirpet, Turkapally Village, Medchal District, Hyderabad 500 101 Telangana, India; 67Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 68Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 69Department of Chemistry, University of California, One Shields Ave, Davis, California 95616; 70Horva´ th Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem ter 1, Hungary; 71Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem ut 10, Hungary; 72Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way Newark, Delaware 19711; 73Proteomics Core Facility, University of Gothenburg, Medicinaregatan 1G SE 41390 Gothenburg, Sweden; 74Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Institute of Biomedicine, Sahlgrenska Academy, Medicinaregatan 9A, Box 440, 405 30, Gothenburg, Sweden; 75Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Bruna Straket 16, 41345 Gothenburg, Sweden; 76Department of Chemistry, University of Hamburg, Martin Luther King Pl. 6 20146 Hamburg, Germany; 77Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; 78Laboratory of Mass Spectrometry of Interactions and Systems, University of Strasbourg, UMR Unistra-CNRS 7140, France; 79Natural and Medical Sciences Institute, University of Tu¨ bingen, Markwiesenstrae 55, 72770 Reutlingen, Germany; 80Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 81Division of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 82Department of Chemistry, Waters Corporation, 34 Maple Street Milford, Massachusetts 01757; 83Zoetis, 333 Portage St. Kalamazoo, Michigan 49007 Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received July 24, 2019, and in revised form, August 26, 2019 Published, MCP Papers in Press, October 7, 2019, DOI 10.1074/mcp.RA119.001677 ER: NISTmAb Glycosylation Interlaboratory Study 12 Molecular & Cellular Proteomics 19.1 Downloaded from https://www.mcponline.org by guest on January 20, 2020 ted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide communityderived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Molecular & Cellular Proteomics 19: 11–30, 2020. DOI: 10.1074/mcp.RA119.001677.L

Glycan structural determinants and their role in microbial interaction

Thesis by publication."A thesis submitted for the degree of Doctor of Philosophy""April 2013"Includes bibliographical references.1. Introduction - literature review and project rationale -- 2. Comparative structural analysis of the glycosylation of salivary and buccal cell proteins: innate protection against infection by Candida albicans -- 3. Blood group antigen are involved in the interaction of C. albicans with buccal epithelial cells -- 4. Structural feature ions for distinguishing N- and O-linked glycan isomers by LC-ESI-IT MS/MS -- 5. Tandem mass spectra of glycan substructures enables the multistage mass spectrometric identification of determinants on oligosaccharides -- 6. Summary and future directions.Microbial infection is initiated only after adherence to the host cell surface. In many cases the microbial interactions with the host surface are mediated between the glycans on the host cell surface and the carbohydrate binding proteins of the pathogen. Mucosa! epithelial surfaces, such as coat the oral cavity, are potential sites for colonisation by oral microorganisms. Saliva constantly bathes the buccal epithelial cell (BEC) surface of the mouth and in this study we have used the oral cavity as a model system to demonstrate the innate immune protective role played by the glycan receptors on the proteins of saliva against the adhesion of the oral pathogen C. a/bicans to the BEC. Thereby, this work could help design glycan inhibitors similar to the host's evolved innate immune system to naturally evade pathogens and thus address the problem of increased microbial resistance to antibiotics. In the first phase of this work (Chapter 2, Publication I), a flow cytometry based adhesion assay was developed to quantify the interaction between buccal cells and the commensal oral pathogen Candida albicans. The structures of the N-and 0-linked oligosaccharides on the glycoproteins of saliva and BEC membranes were analysed using capillary carbon negative ion LC-ESI MS/MS. A total of 190 glycan structures were characterised and found to be qualitatively similar between saliva and epithelial buccal cell membrane proteins, but differed quantitatively in their relative amounts. The similarity of the terminal glycan epitope structures on saliva and BEC membrane glycoproteins, and the fact that whole saliva and released glycans from salivary proteins inhibited the interaction of C. albicans with BEC, confirmed the protective role of the glycans on salivary glycoproteins against pathogen infection of the oral surface mucosa. Further investigation of the glycan determinants identified on the terminal N-and 0-glycan structures of BEC and saliva supported the proposed function of blood group antigens as an evolutionary selection against pathogen infection. The detailed mass spectrometric glycan characterisation and relative quantification of BEC membrane glycans (Chapter 3) was carried out on 19 individuals of various A, 8, AB and O blood group types. The N-glycans of BEC were similar in all secretor individuals and did not display the A and B determinants; while non-secretors did not possess the 0/H antigens. Jn contrast, the 0-glycans on the membrane proteins of BEC from secretory individuals expressed the A, B and H antigens, while the non-secretors lacked any of these structures. The Lewis x/a and Lewis y/b blood group antigens were observed on secretor individuals N- and O-glycans; in non-secretor individuals, as expected, only Lewis x/a antigens were present. Multivariate statistical analysis showed that C. albicans demonstrated a significantly (p < 0.05) higher preference to adhere to BEC of blood group O individuals. The diagnostic and signature fragment ions produced by negative ion MS/MS fragmentation, together with the elution selectivity of PGC retention, were identified and applied to differentiate the N- and 0-glycan isomer structures of the complex salivary glycans (Chapter 4, Publication 2). This labour-intensive approach led to the construction of a PGC-LC-ESI-IT-MS2 tandem mass spectral repository on the online UniCarb-DB database which was further expanded by an online MS2 fragment spectral library of 30 common glycan substructures that typically occur at the non-reducing terminus of glycoconjugates, fragmented in the positive and negative ion mode (Chapter 5, Publication 3). The substructure spectra were used to identify and confirm terminal glycan determinants from the multistage (MS3) mass spectra of the salivary N- and O-glycans. These mass spectrometric insights will enable the easier identification and confirmation of glycan determinants on oligosaccharides released from glycoproteins in future analyses. The work presented here applies negative ion PGC-LC-ESI-MS/MS analysis for the detailed characterisation of the N- and O-glycans on epithelial cell surface and secreted fluid proteins and demonstrates the role played by terminal glycan structural determinants as receptors for pathogen binding.Mode of access: World wide web1 online resource (vii, 192 pages) illustration

Deciphering the Importance of Glycosphingolipids on Cellular and Molecular Mechanisms Associated with Epithelial-to-Mesenchymal Transition in Cancer

Every living cell is covered with a dense and complex layer of glycans on the cell surface, which have important functions in the interaction between cells and their environment. Glycosphingolipids (GSLs) are glycans linked to lipid molecules that together with sphingolipids, sterols, and proteins form plasma membrane lipid rafts that contribute to membrane integrity and provide specific recognition sites. GSLs are subdivided into three major series (globo-, ganglio-, and neolacto-series) and are synthesized in a non-template driven process by enzymes localized in the ER and Golgi apparatus. Altered glycosylation of lipids are known to be involved in tumor development and metastasis. Metastasis is frequently linked with reversible epithelial-to-mesenchymal transition (EMT), a process involved in tumor progression, and the formation of new distant metastatic sites (mesenchymal-to-epithelial transition or MET). On a single cell basis, cancer cells lose their epithelial features to gain mesenchymal characteristics via mechanisms influenced by the composition of the GSLs on the cell surface. Here, we summarize the literature on GSLs in the context of reversible and cancer-associated EMT and discuss how the modification of GSLs at the cell surface may promote this process

A Platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells

Rationale: Glycosphingolipids (GSLs) constitute a highly diverse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, analytical technologies. Methods: Neutral and acidic GSL standards were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymatically released using endoglycoceramidase II (EGCase II), separated by porous graphitized carbon (PGC) liquid chromatography and structurally characterized by negative ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (10⁷ cells) samples. Results: Glycans were released from GSL standards comprising of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 µg of GSL. The platform of analysis was then applied to GSLs isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatographically resolved on PGC and characterized by comparison with the MS² fragment ion spectra of the glycan standards and by application of known structural MS² fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures containing Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides. Conclusion: We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS²fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs.17 page(s

Sialyl Lewis X conjugated nanodiamonds for vascular targeting

Arteriovenous malformations (AVMs) of the brain are congenital lesions that are the major causes of haemorrhagic stroke in young adults. Most small AVMs are curable while a majority of the large lesions are difficult to cure using currently available techniques. Novel therapeutic strategies such as vascular targeting are attractive options for the treatment of these large lesions by selective occlusion of the AVM vessels. This targeted approach requires specific endothelial surface markers that are highly discriminatory between normal and AVM vessels. Although AVM vessels differ from normal vessels morphologically, there is a lack of specific markers that could be used as targeting agents. This model system has potential for vascular targeting based therapy of AVM's. Sialyl Lewis X conjugated nanodiamonds could be eventually used as drug delivery vehicles for prothrombotic agents that can block the AVM vessels and eliminate the risk of haemorrhagic stroke. Targeted delivery of drugs and imaging probes to the selectins on irradiated endothelium, or to inflammation sites, holds promise to improve management and treatment of many diseases.1 page(s

FUT1 genetic variants impact protein glycosylation of porcine intestinal mucosa

A massive use of antibiotics in industrial pig production is a major cause of the rapidly rising bacterial resistance to antibiotics. An enhanced understanding of infectious diseases and of host-micr obe interactions has the potential to explore alternative ways to improve pig health and reduce the need for antibiotics. Host-microbe interactions depend on host-expressed glycans and microbe-carrying lectins. In this study, a G > A (nucleotide 307) missense mutation in the porcine α1,2fucosyltransferase 1 gene (FUT1), which has been reported to prevent infections by the common porcine enteric pathogen F18 fimbriated Escherichia coli, provided a unique opportunity to study glycan structures potentially involved in intestinal infections. N- and O-Linked glycans of the intestinal mucosa proteins were characterized in detail using LC-MS/MS. Relative abundances of all glycans were determined and compared between four heterozygous pigs (FUT1-307A/G) and four age-matched homozygous pigs from the same 2 litters carrying the missense FUT1 gene constellation (FUT1-307A/A). None of the characterized 48 N-linked glycans was found to be regulated by the FUT1 missense mutation, while 11 of the O-linked glycans showed significantly altered abundances between the two genotypes. The overall abundance of H-antigen carrying structures was decreased fivefold, while H-antigen precursors and sialylated structures were relatively more abundant in pigs with the FUT1 missense mutation. These results provide insight into the role of FUT1 on intestinal glycosylation, improve our understanding of how variation in FUT1 can modulate host-microbe interactions, and suggest that the FUT1 genetic variant may help to improve pig gut health.16 page(s

Assessing the role of pharyngeal cell surface glycans in group a streptococcus biofilm formation

2020, MDPI AG. All rights reserved. Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20-40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion

Tissue glycomics distinguish tumour sites in women with advanced serous adenocarcinoma

In the era of precision medicine, the tailoring of cancer treatment is increasingly important as we transition from organ-based diagnosis towards a more comprehensive and patient-centric molecular diagnosis. This is particularly the case for high-grade serous adenocarcinomas of the ovary and peritoneum, which are commonly diagnosed at an advanced stage, and collectively treated and managed similarly. We characterized the N- and O-glycome of serous ovarian (OC) and peritoneal cancer (PC) tissues using PGC-LC-ESI-IT-MS/MS profiling and validated the discriminatory glycans and their corresponding glyco-gene expression levels using cell lines and transcriptomic data from 232 patients. Overall, the N- and O-glycan repertoires of both cancer types were found to comprise mostly of α2,6-sialylated glycan structures, with the majority of N-glycans displaying the biantennary mono- and disialylation as well as bisecting-type biantennary glycans. The MS profiling by PGC-LC also revealed several glycan structural isomers that corresponded to LacdiNAc-type (GalNAcβ1-4GlcNAc) motifs that were unique to the serous ovarian cancers and that correlated with elevated gene expression of B4GALNT3 and B4GALNT4 in patients with serous cancer. Statistical evaluation of the discriminatory glycans also revealed 13 N- and 3 O-glycans (P < 0.05) that significantly discriminated tumour-sampling sites, with LacdiNAc-type N-glycans (m/z 1205.02- and m/z 1059.42- ) being associated with ovarian-derived cancer tissue and bisecting GlcNAc-type (m/z 994.92- ) and branched N-glycans (m/z 1294.02- and m/z 1148.42- ) upregulated at the metastatic sites. Hence, we demonstrate for the first time that OC and PC display distinct molecular signatures at both their glycomic and transcriptomic levels. These signatures may have potential utility for the development of accurate diagnosis and personalized treatments

Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines : glycan structures reflect gene expression and DNA methylation status

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 α 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.20 page(s