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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, Vol. 90, No. 13, 03.07.2018, p. 7871-7879.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Costa, J, Gatermann, M, Nimtz, M, Kandzia, S, Glatzel, M & Conradt, HS 2018, 'N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines', ANAL CHEM, vol. 90, no. 13, pp. 7871-7879. https://doi.org/10.1021/acs.analchem.7b05455

APA

Costa, J., Gatermann, M., Nimtz, M., Kandzia, S., Glatzel, M., & Conradt, H. S. (2018). N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines. ANAL CHEM, 90(13), 7871-7879. https://doi.org/10.1021/acs.analchem.7b05455

Vancouver

Costa J, Gatermann M, Nimtz M, Kandzia S, Glatzel M, Conradt HS. N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines. ANAL CHEM. 2018 Jul 3;90(13):7871-7879. https://doi.org/10.1021/acs.analchem.7b05455

Bibtex

@article{7176924d0cca468bb658113ef8d4b405,
title = "N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma Cell Lines",
abstract = "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.",
keywords = "Journal Article",
author = "J{\'u}lia Costa and Maren Gatermann and Manfred Nimtz and Sebastian Kandzia and Markus Glatzel and Conradt, {Harald S}",
year = "2018",
month = jul,
day = "3",
doi = "10.1021/acs.analchem.7b05455",
language = "English",
volume = "90",
pages = "7871--7879",
journal = "ANAL CHEM",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "13",

}

RIS

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 -