N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma

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N-GLYCOSYLATION OF EXTRACELLULAR VESICLES FROM HEK-293 AND GLIOMA CELL LINES Julia Costa, Maren Gatermann, Manfred Nimtz, Sebastian Kandzia, Markus Glatzel, and Harald S. Conradt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05455 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Analytical Chemistry

Title: N-GLYCOSYLATION OF EXTRACELLULAR VESICLES FROM HEK-293 AND GLIOMA CELL LINES

Authors: Júlia Costa1*, Maren Gatermann2, Manfred Nimtz3, Sebastian Kandzia2, Markus Glatzel4, Harald S. Conradt2 1

Laboratory of Glycobiology, Instituto de Tecnologia Química e Biológica António

Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal 2

GlycoThera GmbH, Feodor-Lynen Strasse 35, D-30625 Hannover, Germany

3

Helmholtz-Zentrum für Infektionsforschung, D-38124 Braunschweig, Germany

4

Institute of Neuropathology, University Medical Center Hamburg-Eppendorf (UKE),

Martinistrasse 52, D-20246 Hamburg, Germany *To whom correspondence should be addressed: Dr. Júlia Costa Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal E-mail: [email protected] Phone: +351214469437 Fax: +351214411277

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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. Nglycans were released from membrane-derived tryptic glycopeptides with peptide Nglycosidase F, labelled with 2-aminobenzamide and analysed by NP-HPLC and MALDI-TOF mass spectrometry. For the three cell lines, enrichment in complex Nglycans 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.


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INTRODUCTION Extracellular vesicles (EVs) are released by cells and have plasma membrane (microvesicles) or multivesicular endosome (exosomes) origins. EVs carry intracellular material from inside the cell to the extracellular environment and are found in bodily fluids, such as blood or cerebrospinal fluid. They are also internalized by other cells and, therefore, constitute vehicles for the transfer of biomolecules from cell to cell. EVs have characteristic molecular compositions that result from specific sorting/enrichment mechanisms and are also related with the parent cells. EVs contain specific nucleic acids, proteins and lipids1. There are databases displaying the composition of EVs from various sources: EVpedia (http://evpedia.info/)2; Vesiclepedia (http://www.microvesicles.org)3; Exocarta (http://www.exocarta.org)4. Proteins from the endosomal sorting complex required for transport (ESCRT)-0, -I, -II, -III and the associated proteins are found in EVs since they participate in endosomal membrane budding that occurs during intravesicle biogenesis and multivesicular endosome formation1. In this context proteins commonly used as exosome markers are Tsg101 or Alix. Furthermore, evidence indicates that mono-ubiquitination is important for the sorting of membrane bound proteins to multivesicular endosomes through binding the ESCRT machinery5. Tetraspanins are another class of membrane proteins, which are palmitoylated and may be glycosylated, and that are enriched in EVs and, therefore, are used as EV markers, such as CD9, CD63 or CD816. Proteins that are involved in membrane fusion and transport, such as RabGTPases, annexins or flotillin, are also found in exosomes1. EVs display specific glycoconjugate composition and characteristic glycosignatures7,8. Initial results showed the presence of distinct PrP glycoforms in exosomes9. Microvesicles from T-cells10, skin cancer and colon cancer cell lines11 had specific glycosylation as evaluated by lectin array technology. In ovarian cancer cell lines specific glycosylation profiles were also found in EVs using lectin blotting, NPHPLC and mass spectrometry12-14. Glycans from EVs were suggested as possible candidates for glycoprotein sorting into exosomes via interaction with cellular lectins15. Furthermore, glycans from the surface of exosomes could participate in the interaction with recipient cells, as was observed for α2,3-linked sialic acid from B-cell exosome surface that mediated the binding to sialoadhesin from macrophages and lymph node16. EVs also contain proteoglycans such as syndecan heparan sulfate which has been proposed to have a role in glycoprotein sorting to exosomes17; on the other hand, heparan sulfate glycans from receptor cells are important for their interaction with exosomes18. 3 ACS Paragon Plus Environment


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EVs present in bodily fluids such as urine, blood, cerebrospinal fluid are carriers of cellular molecules to the environment and, therefore, may provide new diagnostic targets. Analysis of urine EVs has been performed by several groups19-23 and highmannose, paucimannosidic and complex N-glycans as well as O-glycans were characterized. In galactosemia patients, glycan composition of urine EVs had a shift from high mannose to complex type, which was not observed in the urinary TammHorsfall glycoprotein; thus, it provided with a potentially interesting diagnostic tool20. Changes in glycosylation have been observed in disease, for example, altered glycosylation is a hallmark of different types of cancer24. Furthermore, EVs have been explored as biomarkers for cancer diagnosis25 and glycoconjugates/glycosylation from EVs constitute emerging targets as cancer biomarkers7. For example, in breast cancer a highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) was found as a marker for proinvasive EVs from tumor cells26. Furthermore, several cancer biomarkers currently used in the clinics have been detected in EVs7. Characterization of glycans from EVs is also useful in the establishment of EV purification protocols based on lectin affinity, which have been used with success27. Detailed structure analyses of the glycans present in EVs has a valuable impact in the investigation of biological roles of glycans and as diagnostic tools. In this paper we present an analytical strategy based on the combination of 2-AB-labelled N-glycan analysis by NP-HPLC with exoglycosidase digestion in conjunction with MALDI-TOF mass spectrometry and lectin blotting, for the detailed elucidation of glycan profiles and structures of EVs from mammalian including glioma cells. EXPERIMENTAL SECTION Cell lines. Human embryonic kidney 293 (HEK-293) cell line was purchased from ECACC (reference 85120602), human H4 neuroglioma cell line was purchased from ATCC (reference ATCC-HTB-148), mouse glioma Tu-2449 cell line has been previously characterized28. Cells were grown in Dulbecco's Modified Eagle Medium high glucose (Sigma), supplemented with 10% fetal bovine serum (Gibco), 100 units/ml penicillin and 0.1 mg/ml streptomycin (Gibco), at 37°C, in 5% CO2. HEK293 and Tu2449 cell lines were authenticated, as authentic 293 and as Mus musculus origin, respectively, by DSMZ, Braunschweig, Germany, at passage number used for EV production for glycan analysis.


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Preparation of extracellular vesicles and total cell membranes.

EVs

were produced from confluent cells in serum-free medium, twice for 48 h. The supernatant was collected and successively centrifuged at 500, 10000 and 100000xg (120000xg for the HEK-293 cells), for 10, 20 and 120 min, respectively, at 4°C, as previously described13. The pellets of the last centrifugations consisted of the EVs fraction; EV protein recovery was in the ranges 74-99, 275-323 and 150-157 µg protein/T175 for HEK-293 (triplicate), H4 and Tu-2449 (duplicate) cells. EVs were also obtained by ultrafiltration of the post-20,000xg supernatant in an Amicon15 filter, cut-off 100 kDa. For the separation of total cell membranes (CMs), confluent cells were incubated with 0.5 M ethylenediamine tetraacetic acid pH 8.0, for 10 min, collected with a cell scraper and centrifuged at 500xg, 5 min. Then, cells were sonicated on ice with 3 cycles of 5 seconds, at 70% power, Branson Digital Sonifier Models 250/450 and 2 min pause in between cycles for cooling. CMs were collected as the pellet of a 100000xg centrifugation, for 1h, as previously described14. EV and CM fractions were resuspended in PBS; aliquots were frozen in liquid nitrogen and kept at -80ºC. Samples were in general thawed only once for analysis. Immunoblotting.

Proteins were analysed by SDS-PAGE and transferred to

polyvinyledene fluoride membranes that were blocked for 1 h with 5% defatted dry milk (Nestle) in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST). The following antibodies were used: mouse anti-CD63 (1:500) (ThermoFisher), mouse anti-CD9 (1:250) (BD), rabbit anti-CD9 (1:500) (Sigma), goat anti-human LGALS3BP (1:2000) (R&D), goat anti-Tsg101 (1:1000) (ThermoFisher), mouse anti-annexin-I (1:5000) (BD), mouse anti-Alix (1:1000) (ThermoFisher), rabbit anti-CD81 (1:1000) (Sigma), mouse anti-flotillin-2 (1:1000) (BD). Secondary antibodies were sheep anti-mouse IgG coupled to HRP (1:4000), donkey anti-rabbit IgG coupled to HRP (GE Healthcare), rabbit antigoat IgG coupled to HRP (1:20000) (Sigma). Washings were with TBST. Detection was performed with the Immobilon Western chemiluminescent HRP substrate (Millipore). CD63, CD9 and CD81 were analysed in non-reducing conditions. Electron microscopy. For negative staining, EV and CM fractions were fixed in 2% formaldehyde in PBS overnight at 4ºC. EVs were added to 200 mesh grids (Ted Pella 01800-F) coated with 1% (w/v) formvar in chloroform and carbon and further fixed with 1% (v/v) glutaraldehyde in PBS for 5 minutes. The samples were stained with a uranyl-oxalate solution pH 7 for 5 minutes and methyl-cellulose-uranyl acetate for 10 minutes at 4ºC.



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For immunocytochemistry, EV or CM samples, at 0.6mg/ml protein concentration, were added to mesh grids 100 (coated with 1% Formvar, carbon and glow discharged) and fixed with 4% paraformaldehyde in PBS. Blocking was done with 1% BSA IgG free (Sigma, A-2058) in PBS. Grids were then incubated with primary (anti-CD9 1:25; anti-CD63 1:50; anti-LGALS3BP 1:200) and secondary (12 nm colloidal gold labelled goat anti-mouse IgG 1:20 and donkey anti-goat IgG 1:20; Jackson ImmunoResearch) antibodies in blocking buffer, followed by fixation with 1% glutaraldehyde in PBS and staining with 2 % uranyl acetate. Grids were observed at 100 kV on a Hitachi-7650 Transmission Electron Microscope and photographed using an AMT XR41-M Mid-mount camera. N-glycan isolation and analysis of 2-aminobenzamide labeled N-glycans by NP-HPLC. N-Glycans were released from EV and CM tryptic glycopeptides (corresponding to 250-350 µg protein) with peptide N-glycosidase F (PNGase F; Prozyme GKE-5006b 2.5 U/ml). Purified N-glycans were then labeled with 2aminobenzamide (2-AB) using a GMP validated derivatisation procedure based on the previously published method of Bigge 29. The detailed procedures are given in Supporting Information, Material and Methods. Control for sialic acid stability during 2AB-labeling was performed using tri- and tetrasialylated (α2,6-sialylated) N-glycans from bovine fetuin as well as α2,3-sialylated standard N-glycans. Mapping of 2-AB-labeled glycans was done with an ACQUITY UPLC BEH glycan column from Waters Corporation (Milford, MA, USA). An HPLC system from Dionex (ThermoFisher Scientific) with an Ultimate 3000 RS Fluorescence detector FLD-3400RS was used. Mobile phase A was 85 % (v/v) acetonitrile and mobile phase B was 250 mM ammonium formate pH 4.4, at a flow rate of 0.40 ml/min and at a column temperature of 60°C. Detection was at excitation wavelength = 347 nm, emission wavelength = 420 nm. The gradient was as follows: starting conditions 88% A and 12% B; a gradient from 1-25 min to 82% A; gradient for 20 min to 70% A; gradient for 10 min to 20% A; increase to 88.0 % A and re-equilibration of the column isocratically for 10 min. 2-AB-Labeled reference oligosaccharide standards of known structure were run within each analytical sample sequence (Table S1); they consisted of a large array of high mannose and complex N-glycan structures characterized according to GMP qualification tests that are from a glycan library of the company containing more than 250 N-glycan structures. The amount applied per each NP-HPLC run was obtained typically from 5 to 18 µg EV protein. For structure elucidation/confirmation 2-AB-labelled N-glycans were digested with the following exoglycosidases of known specificity: α(2-3)-sialidase (Salmonella 6 ACS Paragon Plus Environment

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typhirium LT2 expressed in Escherichia coli EC 3.2.1.18, Takara); α(2-3,6)-sialidase (Arthrobacter ureafaciens EC 3.2.1.18, Roche); α-L-(1-3,4)-fucosidase (almond meal EC 3.2.1.111, Prozyme); α-L-(1-2,3,4,6)-fucosidase (bovine kidney EC 3.2.1.51, Sigma); β(1-3,4)-galactosidase (bovine testes, Prozyme); α-galactosidase (green coffee beans EC 3.2.1.22, Sigma). The detailed procedures are presented in Supporting Information, Material and Methods. Quantitative analysis of NP-HPLC chromatograms was done using the Chromeleon 6.8 software. Linearity in the working range was confirmed using 2-ABlabelled high mannose standards Man5GlcNAc2, Man6GlcNAc 2, Man7GlcNAc 2, Man8GlcNAc 2 and Man9GlcNAc 2, and 2-AB-labelled paucimannose standards Man3GlcNAc2 and Man3GlcNAc2Fuc. Peaks were selected manually. Peak areas (C*min) and relative percentages (% in relation to all detected peaks with elution times between 5 and 55 min) were evaluated. For all cell lines the quantification was from two independent EV and CM productions, each one processed for N-glycan analysis, with the exception of Tu-2449 EVs where each production was processed in duplicate for N-glycan analysis. Percentages of more abundant identified peaks are presented as range. The method for quantification of Neu5Gc and Neu5Ac from 2-AB-labeled Nglycans is presented in Supporting Information, Material and Methods. Analysis of desialylated 2-AB labelled N-glycans by MALDI-TOF MS. Desialylated N-glycans with Arthrobacter ureafaciens were analysed with a Bruker ULTRAFLEX time-of-flight (TOF/TOF) instrument as previously described. Mass spectra were recorded in the reflector and positive ion mode. Samples of 1 µl at an approximate concentration of 5 pmol/µl were mixed with equal amounts of the matrix 2,5-dihydroxybenzoic acid. The mixture was spotted onto a stainless steel target and dried at room temperature before analysis. Structures were tentatively assigned to various N-glycans on the basis of their observed masses in comparison to the calculated masses of known N-glycans. Further annotation of N-glycans and database searches were performed using the GlycoWorkbench software30. RESULTS AND DISCUSSION Production, purification and characterization of EVs and CMs from HEK293, H4 and Tu-2449 cells. For EV purification cell supernatants were first centrifuged at 500xg and 10,000xg. This supernatant was then ultracentrifuged at 100,000xg for human neuroglioma H4 and mouse glioma Tu-2449 cells and at 120,000xg for HEK7 ACS Paragon Plus Environment


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293 cells; the pellets corresponded to the EV fraction. The centrifugation speed was higher for HEK-293 cells because material staining positive for EV markers (CD63, CD81, CD9) was detected in the pellet of a 120,000xg centrifugation of the post100,000xg supernatant. Total cell membranes (CMs) were obtained by sonication of the corresponding cells and ultracentrifugation. For EV characterization immunoblotting using antibodies against well-established EV markers (tetraspanins CD63, CD9 and CD81; biogenesis related proteins Tsg101 and Alix; flotillin-2 and annexin-1) was performed, and compared with CM fractions (Figure 1A). Tetraspanins CD63, CD9 and CD81 were detected in HEK-293 and H4 cells. In Tu-2449 cells immunoblotting for CD9 and CD81 showed EV-enriched bands at the expected molecular mass; however, CD81 appeared predominantly at a higher mass than in the human cell lines possibly due to oligomerization or interaction with other proteins in the mouse cells. Tsg101 and Alix were detected in HEK-293 and Tu-2449 EVs. Flotillin-2 was found in EVs from all cell lines, and annexin-1 was detected in EVs from HEK-293 and H4 cells. Calnexin an endoplasmic reticulum marker was enriched in CMs and depleted from EVs, which indicated that EVs were not significantly contaminated with endoplasmic reticulum membranes. EV fractions from the three cell lines were also characterized by electron microscopy and vesicles with the characteristic cup shape and diameters around 100 nm were detected (Figure 1B; Figure S1A). Furthermore, vesicles of smaller as well as larger sizes were also detected in the 100,000xg pellet. On the other hand, CM fractions consisted of vesicles of different sizes and many large membrane aggregates were detected (Figure S1B). In addition, EV markers CD9 and CD63 were visualized on the EV fractions by immunocytochemistry. CD9 and CD63 were present at the surface of cup-shape vesicles with diameters around 100 nm but also in smaller vesicles (Figure 1B, arrowheads). On the other hand, non-vesicular material was also detected. The glycoprotein galectin-3-binding protein (LGALS3BP), previously found associated with EVs from ovarian carcinoma cells13,14, was also strongly detected here in the EV fraction, particularly in the human cell lines. LGALS3BP was predominantly detected at the surface of smaller vesicles/structures of irregular shape (Figure 1B, arrowheads). To investigate if LGALS3BP would also be present in EVs prepared by a distinct method, EVs from HEK-293 cells were approximately 240-fold concentrated by ultrafiltration through a 100 kDa cut-off filter (Figure S2A). Ultrafiltered EVs displayed the markers CD63, CD9, CD81, flotillin-2 and annexin-1 and LGALS3BP was also enriched in this EV fraction (Figure S2A). Here LGALS3BP was also found at the surface of small vesicles/structures of irregular shape (Figure S2B). In agreement 8 ACS Paragon Plus Environment

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LGALS3BP was recently identified in nanoparticles termed exomeres of about 35 nm from several tumor cells31. EVs obtained by ultracentrifugation, the most common method for EV preparation, contained a large set of EV markers for the three cell lines studied detected by immunoblotting, they were deployed of the endoplasmic reticulum marker calnexin and they were visualized by electron microscopy where some EV markers were also detected by immunocytochemistry; therefore, in spite of the presence of nonvesicular material detected by electron microscopy, the fraction obtained was enriched in EVs that essentially obeyed the minimal experimental requirements for definition of extracellular vesicles 32. Thus, they have been used for N-glycosylation analysis. NP-HPLC analysis of 2-AB-labelled N-glycans from EVs. Glycoproteins from CMs and EVs were precipitated with ethanol, solubilized in urea and further digested with trypsin. N-glycans were released from tryptic glycopeptides with peptide Nglycosidase F and were fluorescently labelled with 2-AB at the reducing terminus for increased sensitivity, which also allowed further quantitative analysis after chromatographic separation29. Labelled glycans were analysed by NP-HPLC and the gradients used allowed a high level of resolution (Figure 2A, B, C, second panels). EVs from HEK-293 cells were enriched in sialylated glycans as evaluated from peak disappearance after digestion with α2,3- and α2,3/6-sialidases (Figure 2A, third and forth panels). Sialic acid was predominantly in α2,3-linkage whereas α2,6-linked sialic acid was only present to a minor extent (Figure 2A). These results are in agreement with the lectin blots with Maackia amurensis lectin (binds Neu5Ac/Gcα2,3Galβ1,4GlcNAcβ1-R) and Sambucus nigra agglutinin (binds Neu5Acα2,6Gal/GalNAc-R) (Figure S3). Predominant sialylated peaks were identified as tetraantennary proximally fucosylated glycans with two (3.63.9%), three (3.5-3.9%) and four (1.9-2.3%) Neu5Ac in α2,3-linkage from comigration with reference standards (indicated with *, ** and *** in Fig. 2A, second panel). After desialylation complex N-glycans of the di- (5.9-6.7%), tri- (7.5-8.1%) and tetraantennary (17.7-20.0%) type with proximal fucose were identified (Figure 2A, forth panel) by comigration with reference standards (Figure 2A, sixth panel). So, the tetraantennary N-glycan with proximal fucose was the predominant species. In agreement, fucosylated structures were also identified by lectin blotting with the lectin from Aleuria aurantia (Figure S3). Concerning H4 glioma cells, EV N-glycans contained predominantly α2,3-linked sialic acid and also a minor amount of α2,6-linked sialic acid (Figure 2B). Predominant



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sialylated peaks consisted of tetraantennary proximally fucosylated glycans with two (6.3-6.7%), three (7.1-7.8%) and four (7.9-8.5%) Neu5Ac in α2,3-linkage from comigration with reference standards (shown in Fig. 2B, second panel). In agreement, the predominant N-glycan in H4 EVs after desialylation was a tetrantennary complex glycan with proximal fucose (26.9-28.9%) (Figure 2B). Other studies have shown that gliomas overexpress terminal sialic acids compared to human brain controls, and α2,3was found to be predominant over α2,6-linked sialic in gliomas33. However, studies on the detailed structure analysis of glioma glycosylation and particularly N-glycosylation are scarce. N-glycans from Tu-2449 EVs also had α2,3- and α2,6-linked sialic acid (Figure 2C, third and forth panels). Comigration with glycan standards indicated the presence of tetraantennary proximally fucosylated glycans with two, three and four Neu5Ac in α2,3-linkage (Fig. 2C, second panel). After desialylation complex glycans of the di-, triand tetrantennary type were detected (Figure 2C forth panel). Contrary to that observed for the two human cell lines a high level of heterogeneity was observed after desialylation. This was partly due to the presence of peripheral Galα3Gal as supported by comigration with standards (Fig. 2C, forth panel) and degalactosylation with αgalactosidase. The presence of α-galactose-containing structures are commonly found in murine cell lines as described in other studies34, and absent from human cell lines as presented in this study. We also found N-glycolylneuraminic (Neu5Gc) in EVs as detected by HPAECPAD analysis. Calculated percentages of Neu5Gc/(Neu5Gc+Neu5Ac) for HEK-293, H4 and Tu-2449 EVs were: 1.8-2.4, 0.7-1.1 and 0.6-0.8%, respectively. Neu5Gc was also detected in the CM fractions of the three cell lines. HEK-293 and H4 EVs are of human origin and should not contain endogenous Neu5Gc since it is known that humans lack endogenous Neu5Gc due to an inactive CMAH gene35. Therefore, the presence of Neu5Gc in HEK-293 and H4 EVs originated from bovine serum since cells were grown in the presence of 10% bovine serum prior to EV production (only production was done in serum free medium). This is in agreement with reports for recombinant proteins produced from human cells in the presence of animal sera and/or animal-derived media additives used in the production process35. On the other hand, Tu-2449 EVs being of mouse origin would not be expected to have comparatively lower levels of Neu5Gc in view of the reported presence of endogenous Neu5Gc in mouse cells. This was probably due to the fact that Tu-2449 cells are derived from brain tissue (glioma), and vertebrate brains have very low amounts of Neu5Gc, which is mostly expressed on the endothelial lining of blood vessels36. The comparatively lower amounts of Neu5Gc 10 ACS Paragon Plus Environment

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could also be due to the competitive presence of terminal Galα3Gal structures or to the cell metabolism. The presence of Galα3Gal and Neu5Gc-containing glycans in EVs is a very relevant issue to be considered when using EVs obtained from cultured cells for therapy (e.g, delivery or vaccination), since humans produce antibodies against these structures and there is the risk of an immunogenic reaction against non-human glycan epitopes present on EVs, similarly to that described for recombinant biotherapeutics. MALDI-TOF MS analysis of desialylated 2-AB-labelled N-glycans from EVs. N-glycans derivatized with 2-AB were analysed by MALDI-TOF MS after desialylation with A. ureafaciens sialidase (Figure 3). Removal of capping sialic acid allowed reducing the complexity of the 2-AB-labelled glycan profile due to different isomeric structures (e.g. sialylation at different antenna). For HEK-293 EVs the major peaks observed were of the complex di-, tri- and tetraantennary type with proximal fucose with monoisotopic masses of 1929.7, 2294.8 and 2660.0, respectively (Figure 3A, second panel; Table S2); this was in agreement with the observation by NP-HPLC (previous section). Peaks at m/z 1970.7, 2335.9, 2701.0 with mass increment of 41 Dalton (Figure 3A, second panel; Table S2) were compatible with the corresponding LacdiNAc (GalNAcβ4GlcNAc)-containing structures. In agreement, lectin blotting analysis with the WFA lectin (recognizes N-acetylgalactosamine) 37 showed the presence of glycoproteins containing terminal GalNAc (Figure S3). These findings are in agreement with other reports where LacdiNAc-containing structures were identified in HEK-293 cells, for example, in membrane glycoproteins38 or in recombinant factor VII39. For H4 cells the major peaks detected at m/z 1929.7, 2294.8 and 2660.0 were compatible with complex N-glycans of the di,-, tri-, and tetraantennary type with proximal fucose (Figure 3B, second panel; Table S3), which was in agreement with the findings by NP-HPLC analysis (previous section). For Tu-2449 EVs peaks with m/z values compatible with di-, tri- and tetraantennary glycans with proximal fucose were also observed (Figure 3C, second panel). Furthermore, intense peaks at m/z 1945.7, 2091.8, 2107.8, 2456.9, 2618.9, 2822.0, 2984.1 and 3146.1 (Figure 3C, second panel; Table S4) were compatible with N-glycans of the complex-type with peripheral α3-linked galactose, which was in agreement with the NP-HPLC profiles (Figure 2C, second panel).



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Comparison of EV and CM profiles. The N-glycan profiles of CMs from all cell lines were very distinct from the respective EV profiles. Although the level of heterogeneity was high, very intense peaks could be identified that were common to the three cell lines. These intense peaks consisted of high mannose glycans Man5GlcNAc2 (M5), Man6GlcNAc2 (M6), Man7GlcNAc2 (M7), Man8GlcNAc2 (M8) and Man9GlcNAc2 (M9) as evaluated by co-migration with authentic high mannose reference standards (Figure 2A, B, C, first panel) and corroborated by peak disappearance after digestion with α-mannosidase (HEK-293 CMs, Figure S4; H4 CMs, Figure S5; Tu-2449 CMs, Figure S6). Peaks co-eluting with high mannose glycans constituted 45.8-47.8, 44.8-44.9 and 38.9-40.3% of the total glycans for HEK293, H4 and Tu-2449 CMs, respectively. The presence of the high mannose series was also detected by MALDI-TOF MS analysis (Figure 3A, B, C; Tables S2, S3, S4) and by HPAEC-PAD analysis for Tu-2449 and H4 CMs (results not shown). Since the CM fraction represents total cellular membranes it contains the endoplasmic reticulum (shown by the detection of calnexin in Figure 1A) where protein N-glycosylation is initiated and which is enriched in high mannose-containing glycoproteins. Although EVs were enriched in complex N-glycans as shown in the two previous sections, high mannose glycans were also detected but in comparatively lower amounts (12.0-12.8, 19.4-21.8 and 17.7-18.7% for HEK-293, H4 and Tu-2449 EVs, respectively) (Figure 2A, B, C, second panel). Previously, we had already found that exosomes from ovarian carcinoma cells although enriched in complex glycans also contained high mannose glycans13. Other studies that compared the N-glycan profiles from the cell surface with those from total membranes of HEK-293 cells also showed the presence of high mannose glycans on the cell surface38. The distinct proportions of the individual glycans between CMs and EVs adds more to the need of investigating if glycans would mediate sorting of glycoproteins into EVs via interaction with intracellular lectins as suggested before15; in this context, modulation of the cellular glycosylation machinery by incubation with inhibitors or through glycosylation engineering would be relevant approaches. Furthermore, the detailed characterization of glycan profiles/structures from plasma membrane or multivesicular endosome membranes from which microvesicles and exosomes originate would be interesting. Changes in glycosylation have been observed in glioma, which include altered composition of specific gangliosides, changes in protein glycosylation and glycosyltransferase expression33. Of relevance, high levels of α2,3-linked sialic acid in glioma cells and brain tumors were found33. Using glioma U-373 MG cells, α2,3sialyltransferase III (ST3Gal III) transfection resulted in increased invasivity, whereas


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α2,6-sialyltransferase (ST6Gal I) expression abolished invasion in vitro and induced alterations in cell morphology, cell-spreading, and adhesion-mediated protein tyrosine phosphorylation; furthermore, ST6Gal I transfectants produced no intracranial tumors in immunodeficient mice, contrary to control parental cells or ST3Gal III-transfected cells40. α3β1 integrin has been suggested to be the sialic acid carrier of relevance in those findings. In the present study we also found the predominance of α2,3-linked sialic acid in H4 human glioma cells. In addition, we found the predominance of high antennarity N-glycans that require the activity of GlcNAc-transferase V for the biosynthesis of the β1,6-branch. This is a relevant issue in several tumor cell types, and particularly, in glioma. For example, stable transfection of GlcNAc-transferase V into human glioma U-373 MG cells resulted in changes in cell morphology and focal adhesions and a marked increase in glioma invasiveness in vitro41. Using the methodologies presented here detailed structure elucidation of Nglycan structures from EVs of HEK-293, H4 and Tu-2449 cells have been characterized for the first time to our knowledge. Furthermore, the information obtained from the NP-HPLC profiles is quantitative. These results provide the basis to further investigate the biological role of N-glycans from EVs for the different cell lines and also to engineer EV surface glycosylation through the action of glycosyltransferases/glycosidases and glycosylation inhibitors. A relevant aspect in this context is further refinement of EV purification by additional techniques in order to remove contaminating proteins not associated with EVs that are known to copurify with them 42. It will also be interesting to perform glycan analysis from EVs prepared using other methodologies such as size exclusion chromatography and to fractionate distinct EV populations (e.g., by sucrose or iodixanol density gradient or immunoisolation), since distinct EV populations may have different biological roles as previously reported 43

. In HEK-293 cells, it would be interesting to investigate if glycans, particularly,

α2,3-linked sialic acid would have a role in the interaction of EVs with other cells as reported for B-cell exosomes binding to sialoadhesin from macrophages and lymph node16; this could have applications in drug delivery since HEK-293 EVs have been largely used in this field44,45. EVs display characteristic glycan profiles, which are nevertheless qualitatively related with the respective parent cells. This aspect is particularly valuable when aiming at biomarker identification. The characterization of human H4 cells provides the basis to further investigate the potential of the glycan structures identified, particularly in conjunction with the respective glycoprotein carriers, as glioma biomarkers. Also



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using these cells it would be possible to develop the investigation on the role of Nglycans, particularly, α2,3-linked sialic acid and tetraantennary proximally fucosylated glycans, in tumorigenicity and invasion, a potentially useful aspect in the development of anti-cancer strategies. In this work N-glycans from the equivalent amount of 250-350 µg EV protein were 2-AB labelled, and the corresponding amount of 5-18 µg EV protein were applied per NP-HPLC run. In view of the observed peak intensities we would predict that reliable analysis could still be performed using about one third of that amount. Therefore, considering the reported values of EV protein that were recovered from blood by size exclusion chromatography (EV proteins from one ml of plasma: 23-49 µg for healthy controls, 30-225 µg for acute myeloid leukaemia patients, 48-84 µg for head and neck squamous cell carcinoma patients)46, from urine by ultracentrifugation (about 300 µg from forty ml urine of healthy individuals)23, and from cerebrospinal fluid by ultracentrifugation (around 2.5-5.5 µg EV proteins from one ml cerebrospinal fluid from several diseased individuals)47, we infer that the analytical strategies presented in this work are suitable for the analysis of EVs from those human bodily fluids with the purpose of biomarker identification.

CONCLUSIONS In this work an analytical strategy based on complementary approaches of NPHPLC, exoglycosidase digestion, MALDI-TOF MS and lectin blotting allowed high resolution quantitative analysis of EVs and CMs glycans from HEK-293 and glioma cell lines. EVs and CMs exhibited very distinct profiles. On one hand, EVs were found to be enriched in complex-type glycans and were qualitatively related with the parent cells. On the other hand, CMs were specifically enriched in high mannose glycans. The results obtained have several valuable applications, including: the development of new tools for EV purification based on lectin affinity; investigation of the role of glycans in glycoprotein sorting to EVs; investigation of the role of glycans from EVs in the interaction with other cells; investigation of the role of glioma glycans on tumorigenesis; identification of potential glioma biomarkers.


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ASSOCIATED CONTENT Supporting Information Available: Material and methods section; figure with electron microscopy of EVs (HEK-293, H4 and Tu-2449 cells) and CMs (HEK-293 and Tu-2449 cells); figure with immunoblotting and immunocytochemistry of HEK-293 EVs obtained by ultrafiltration; figure with lectin blotting of HEK-293 CMs and EVs; figures with NPHPLC analysis and exoglycosidase digestions of CMs HEK-293, H4 and Tu-2449 cells; table with oligosaccharide standards, table with peak quantifications from NP-HPLC analysis; tables with monoisotopic masses, proposed compositions and compatible structures of desialylated 2-AB labelled N-glycans analysed by MALDI-TOF-MS (CMs and EVs from HEK-293, H4 and Tu-2449 cells). This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS We acknowledge: A.L. Sousa, S. Bonucci and E.M. Tranfield from the Electron Microscopy Facility at the Instituto Gulbenkian de Ciência for sample processing and technical expertise; J. Gomes, ITQB NOVA, for technical assistance. This work was supported by Euronanomed 2 ERA-NET, project GlioEx (ENMed/0001/2013), Fundação para a Ciência e a Tecnologia, Portugal; iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia / Ministério da Ciência e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement.



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LEGENDS Figure 1. Characterization of EVs from HEK-293, H4 and Tu-2449 cells. A. Immunoblotting of markers in EV fractions compared to total membrane (CM) fractions. For HEK-293 and Tu-2449 cells 10 µg protein were applied per lane; for H4 cells 3 µg protein were applied per lane. B. Immunocytochemistry of CD9, CD63 and LGALS3BP in EVs from HEK-293 cells. Staining is indicated with arrowheads. Bar: 100 nm. Figure 2. NP-HPLC profiles of 2-AB labelled N-glycans of EV and CM fractions from HEK-293 (A), H4 (B) and Tu-2449 (C) cells. Digestion of total EV N-glycans with exoglycosidases is presented. High mannose and complex type standards are shown. Predominant structures in the CM fractions are of the high mannose type whereas predominant structures in EVs are of the complex type. Relevant assigned structures are displayed close to the corresponding peaks. Representation of glycans is according to the Consortium for Functional Glycomics.Tetraantennary glycans proximally fucosylated with 2, 3 and 4 Neu5Ac are indicated with *, ** and ***, respectively. Figure 3. MALDI-TOF MS profiles of 2-AB labelled N-glycans of EV and CM fractions from HEK-293 (A), H4 (B) and Tu-2449 (C) cells. The m/z values of major peaks corresponding to monoisotopic structures are displayed. Peaks corresponding to high mannose structures Man5GlcNAc2 to Man9GlcNAc2 (M5 to M9) are indicated. Structures of complex-type N-glycans compatible with the measured m/z values are displayed. More complete lists of m/z values of more intense peaks are presented in Table S2 (HEK-293 cells), Table S3 (H4 cells) and Table S4 (Tu-2449 cells).


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ACS Paragon Plus Environment


Figure 2


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Figure 3



Graphic abstract 397x229mm (72 x 72 DPI)


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N-Glycosylation of Extracellular Vesicles from HEK-293 and Glioma

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