N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines
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N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines. / Costa, Júlia; Gatermann, Maren; Nimtz, Manfred; Kandzia, Sebastian; Glatzel, Markus; Conradt, Harald S.
in: ANAL CHEM, Jahrgang 90, Nr. 13, 03.07.2018, S. 7871-7879.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines
AU - Costa, Júlia
AU - Gatermann, Maren
AU - Nimtz, Manfred
AU - Kandzia, Sebastian
AU - Glatzel, Markus
AU - Conradt, Harald S
PY - 2018/7/3
Y1 - 2018/7/3
N2 - Cells release vesicles to the surroundings, the extracellular vesicles (EVs), which may transmit biomolecules to other cells, and are found in bodily fluids, thus constituting emerging biomarker targets. Many studies on EV nucleic acid, lipid, and protein composition are available; however, detailed characterization of protein glycosylation has been less approached. Here, we describe a strategy for high-resolution quantitative profiling and structure elucidation of N-glycans from EV glycoproteins of three cell lines: human HEK-293, human glioma H4 and mouse glioma Tu-2449. EVs have been purified from cell supernatants by ultracentrifugation and compared with total cellular membranes (CMs). CMs and EVs have been characterized by immunoblotting using a panel of EV-specific antibodies, electron microscopy, and immunocytochemistry. N-Glycans were released from membrane-derived tryptic glycopeptides with peptide N-glycosidase F, labeled with 2-aminobenzamide and analyzed by normal phase-high-pressure liquid chromatography (NP-HPLC) and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. For the three cell lines, enrichment in complex N-glycans was found in EVs concomitant to a small amount of high mannose glycans, whereas CMs were highly enriched in high mannose glycans. In HEK-293 and H4 EVs, the predominant N-glycan was tetraantennary proximally fucosylated with α2,3-linked N-acetylneuraminic acid; HEK-293 EVs also contained the LacdiNAc structure. Mouse Tu-2449 EV profiles were very heterogeneous, with di-, tri-, and tetraantennary proximally fucosylated glycans and the presence of peripheral Galα3Gal structure. The results opened novel perspectives to further investigate the roles of glycans in EVs biological properties and may contribute to the biomarker field in glioma.
AB - Cells release vesicles to the surroundings, the extracellular vesicles (EVs), which may transmit biomolecules to other cells, and are found in bodily fluids, thus constituting emerging biomarker targets. Many studies on EV nucleic acid, lipid, and protein composition are available; however, detailed characterization of protein glycosylation has been less approached. Here, we describe a strategy for high-resolution quantitative profiling and structure elucidation of N-glycans from EV glycoproteins of three cell lines: human HEK-293, human glioma H4 and mouse glioma Tu-2449. EVs have been purified from cell supernatants by ultracentrifugation and compared with total cellular membranes (CMs). CMs and EVs have been characterized by immunoblotting using a panel of EV-specific antibodies, electron microscopy, and immunocytochemistry. N-Glycans were released from membrane-derived tryptic glycopeptides with peptide N-glycosidase F, labeled with 2-aminobenzamide and analyzed by normal phase-high-pressure liquid chromatography (NP-HPLC) and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. For the three cell lines, enrichment in complex N-glycans was found in EVs concomitant to a small amount of high mannose glycans, whereas CMs were highly enriched in high mannose glycans. In HEK-293 and H4 EVs, the predominant N-glycan was tetraantennary proximally fucosylated with α2,3-linked N-acetylneuraminic acid; HEK-293 EVs also contained the LacdiNAc structure. Mouse Tu-2449 EV profiles were very heterogeneous, with di-, tri-, and tetraantennary proximally fucosylated glycans and the presence of peripheral Galα3Gal structure. The results opened novel perspectives to further investigate the roles of glycans in EVs biological properties and may contribute to the biomarker field in glioma.
KW - Journal Article
U2 - 10.1021/acs.analchem.7b05455
DO - 10.1021/acs.analchem.7b05455
M3 - SCORING: Journal article
C2 - 29888905
VL - 90
SP - 7871
EP - 7879
JO - ANAL CHEM
JF - ANAL CHEM
SN - 0003-2700
IS - 13
ER -