Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.


Introduction
Exosomes are membrane vesicles between 40-100 nm in diameter that are secreted by various cell types, including tumor cells, neurons, T-cells, dendritic cells and macrophages. In addition, exosomes were also found in physiological fluids including plasma, cerebrospinal fluid, urine and malignant ascites [1,2].
Exosome biogenesis involves the inward budding of endosomes into multivesicular bodies to form intraluminal vesicles that are then released to the extracellular space. The molecular basis of protein sorting during exosomes formation involves a ubiquitin-dependent mechanism but oligomerization or partitioning of protein into lipid raft domains may be involved in the sorting of certain proteins [2]. Exosomes are secreted from the cell by fusion of multivesicular bodies with the plasma membrane.
Among other functions, exosomes were found to be associated with the transmission of pathogenicity among cells, for example, in tumor progression or neurodegenerative diseases, and they stimulate the immune system [3,4]. Exosomes can interact with target cells through ligand-receptor interactions or lipids, and they have been described to fuse with the plasma membrane or to be internalized via receptormediated endocytosis [5,6]. Lectins and their interactions with carbohydrates have also been found to play a role in exosome recognition and uptake by dendritic cells [7] and macrophages [8].
Exosomes have a unique protein, lipid and glycan composition. They contain proteins involved in cellular events including, membrane transport and fusion, biogenesis of multivesicular bodies, tetraspanins, and they are enriched in lipid microdomains [2]. Concerning glycosylation, exosomes were found to contain a conserved glycan signature relatively to parent cell membranes by lectin array technology [9,10]. Furthermore, specific sialic acid-containing glycoproteins were previously found in exosomes from SKOV3 cells [6].
In the present work, we have characterized glycoproteins and N-glycans from ovarian carcinoma exosomes. The sialoglycoprotein galectin-3-binding protein (LGALS3BP) was found to be strongly enriched in exosomes. Furthermore, exosomes had complex and high mannose glycans.
Confluent cells were cultivated for 48 h in serum-free medium. The supernatant was collected and centrifuged at 500, 10,000 and 100,000 x g, 10, 20 and 120 min, respectively, at 4 °C. The pellet of the last centrifugation consisted of crude exosomes, which was further used for exosome purification by sucrose gradient as previously described [5,6]. Essentially, crude exosomes (1 ml suspension) were loaded on top of a stepwise gradient comprising layers of 2 M (1.5 ml), 1.3 M (2.5 ml), 1.16 M (2.5 ml), 0.8 M (2 ml), 0.5 M (2 ml) and 0.25 M (1 ml) sucrose. The gradients were centrifuged for 2.5 h at 100,000xg in a Beckman SW41Ti rotor. Twelve 1 ml fractions were collected from the top (1 to 12) and precipitated with 10 % trichloroacetic acid before immunoblotting or lectin blotting analysis.
The isolation of the microsomal fraction was performed by differential centrifugation. Cells were resuspended in homogenization buffer (1 mM NaHCO 3 , 0.2 mM CaCl 2 , 0.2 mM MgCl 2 , 1mM spermidine, pH 8.0) [14], disrupted with a pellet pestle (Sigma) and centrifuged at 600 x g, 10 min, and 15,000 x g, 10 min. The supernatant was further centrifuged at 100,000 x g, for 120 min, and the pellet consisted of the microsomal fraction.
The isolation of plasma membrane proteins was performed by cell surface biotinylation as previously described [15]. Typically, the following amounts of protein were obtained from cells in one T175: 2 mg for total cell extracts, 0.4 mg for the microsomal fraction, 64 µg for plasma membrane, 36 µg for crude exosomes and 12 µg for exosomes.
Protein concentration was determined by the bicinchoninic acid method.

Lectin blotting, immunoblotting and lectin-affinity chromatography
Proteins were analysed by SDS-PAGE in 10% acrylamide gels. Glycoproteins were detected by lectin blotting after transfer to polyvinylidene fluoride as previously described [13]. Concanavalin A (Con A; Sigma), biotinylated Sambucus nigra agglutinin (SNA) and Maackia amurensis lectin (MAL) (Galab Technologies) were used. As control of Con A specificity, incubation was done in the presence of competitive sugar, 0.2 M methyl-α-D-mannopyranoside. As controls for SNA and MAL specificity exosomes were incubated with sialidases from Vibrio cholerae, Arthrobacter urefaciens and Streptococcus pneumoniae as previously described [6]. Immunoblotting was done as previously described [6] with goat anti-human GALS3BP polyclonal antibody (R&D) and anti-goat immunoglobulin G coupled to horseradish peroxidase (Sigma) as primary and secondary antibodies at 1:2000 and 1:20000 dilutions, respectively. Detection was performed with the Immobilon Western chemiluminescent (Millipore).

Electron microscopy
Preparation of the exosomes for transmission electron microscopy was done using the technique published by Théry et al. [16]. Briefly, centrifuged exosomes were fixed in 2% paraformaldehyde in PBS overnight. Exosomes on 200 mesh carbon coated grids (Ted Pella 01800-F) were further fixed with 1% glutaraldehyde in PBS before being contrasted with uranyloxalate and methy cellulose-UA. Grids were observed at 100 kV on a Hitachi-7650 Transmission Electron Microscope and photographed using an AMT XR41-M Mid-mount camera.

Peptide mass fingerprinting
Gel bands were excised and subjected to in-gel digestion with trypsin according to the protocol previously described [17]. Tryptic digests of individual gel spots were analyzed by capillary liquid chromatography-tandem mass spectrometry. Liquid chromatography was performed using an Ultimate 3000 RSLCnano. Samples were desalted and preconcentrated at a flow rate of 10 µL/ min on a short C18 Nano Trap Column Acclaim PepMap (5 µm, 100 A, 100 µm i.d. x 2 cm; LC-Packings) connected in the front of an analytical capillary column. For the separation with the C18 PepMap column (180 µm i.d. x 15 cm; LC-Packings), a flow rate of 6 µL/min was used. For the chromatography, the following solvents were used: solvent A (H 2 O, 0.1% formic acid) and solvent B (90% acetonitrile, 9.9% H 2 O, 0.1% formic acid). The eluting gradients used for separation of tryptic digestions were, respectively, 1 to 52% B in 103 min, 52 to 99% B in 1 min, followed by 99% B for 10 min and then by 1% B in 5 min. The chromatography system was directly coupled to a three-dimensional highcapacity ion trap (HCTultra ETD II; Bruker Daltonik, Bremen, Germany) mass spectrometer with an electrospray ionization source. Instruments were controlled using DCMS Link (Dionex) and Compass 1.3 (Bruker Daltonics) software. The smart parameter settings (SPS) were for the target mass 600 m/z and for compound stability 100%. The full MS scan mode was standard-enhanced (m/z 300 to 1600). The two most abundant ions detected in each MS scan were selected for collision-induced dissociation (MS/MS) with 1.0 V collision energy. The peptides were analyzed using the data-dependent MS/MS mode over the m/z range 100-3000. Raw spectrum data were processed and Mascot compatible mgf files were created using DataAnalysis 4.1 software (Bruker Daltonik) with the following parameters: compounds threshold 100,000, maximum number of compounds 300, and retention time windows 0.5 min. Searches were performed using BioTools 3.2 software (Bruker Daltonik) together with the algorithm on the Mascot web site (Matrixscience, London, UK) by searching against the NCBI non-redundant human database. Search parameters were set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; mass tolerance, 0.3 Da and MS/MS mass tolerance, 0.3 Da; fixed modification parameter, carbamidomethylation (C).

N-glycans isolation and analysis of 2-aminobenzamide labeled N-glycans by NP-HPLC
Total N-glycans isolation from exosome glycoproteins were performed as previously described [18].
N-glycans were labelled with 2-AB (2-aminobenzamide) according to the derivatisation method of Bigge [19] with reagents from Sigma-Aldrich. After derivatisation, the 2-AB labelled glycan mixtures were purified from excess labelling reagents by gel filtration using PD MiniTrap G-10 size exclusion columns from GE Healthcare. The size exclusion columns were conditioned with 8 ml ultrapure water prior to sample application. The columns were washed with 700 μl ultrapure water followed by elution of 2-AB labelled N-glycans with 500 μl ultrapure water.
Mapping of 2-AB-labelled glycans was done with an ACQUITY UPLC BEH glycan column from Waters Corporation (Milford, MA, USA). An HPLC system from Dionex Corporation (Sunnyvale, CA, USA) 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. Within an analytical sample sequence reference standards were run with various structurally well characterised 2-AB-labelled oligosaccharides.

Analysis of N-glycans by MALDI-TOF MS
Glycans were analysed with a Bruker ULTRAFLEX time-offlight (TOF/TOF) instrument. Mass spectra were recorded in the reflector and positive ion mode. Samples of 1 μl at an approximate concentration of 5 pmol.μl-1 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 Nglycans. Further annotation of N-glycans and database search were performed using the Glyco-Peakfinder platform [20]. Further annotation of N-glycans and database searches were performed using the GlycoWorkbench software [21].

Analysis of 2-AB labeled oligosaccharides by LC-ESI-TRAP-MS
The 2-AB labeled oligosaccharide mixture was analyzed by capillary liquid chromatography-tandem mass spectrometry on the same system used for the peptide analysis but with different parameter settings as follows. Samples were directly injected onto a C18 PepMap column (180 µm i.d. x 15 cm; LC-Packings) at a flow rate of 8 µL/min. The gradient sequence used for chromatographic separation was: 0% B for 5 min; 0% to 35% B in 15 min; 35% to 95% B in 5 min, re-equilibration with isocratic 0% B for 10 min. The smart parameter settings (SPS) were set for the target mass 700 m/z and for compound stability 100 %. The full MS scan mode was standardenhanced (m/z 100 to 2000). The most abundant ion detected in each MS scan was selected for collision-induced dissociation (MS/MS) with 1.4 V collision energy. Ions of an m/z value in the ranges m/z 703.7-704.7 and m/z 771.4-772.4 were preferentially selected. This setup was used for an initial identification of the glycans. Afterwards, a second analysis with the same LC-parameter settings and different MS-settings was performed. In two further runs the target values of m/z 772.4 or 704.2 respectively were isolated and fragmented. Raw spectrum data were then processed using DataAnalysis 4.1 software (Bruker Daltonics). The MS2 fragmentation spectra acquired for each glycan were integrated into one averaged spectrum. Fragmentation analysis was performed with the help of GlycoWorkbench (ver. 2.1) [21].

Identification of sialoglycoproteins specific to exosomes
The microsomal fraction, plasma membrane and crude exosomes from SKOV3 cells have been analysed by lectin blotting with the lectins MAL (binds NeuAcα2,3Galβ1,4GlcNAc/ Glc), SNA (binds NeuAcα2,6Gal/GalNAc) and Con A (binds αmannosyl containing branched glycans predominantly of the high-mannose followed by hybrid-and biantennary complex type). The enriched microsomal fraction was obtained by differential centrifugation (100,000 x g pellet after clarification of the cell extract at 15,000 x g) [22], the plasma membrane enriched fraction was obtained after cell surface protein biotinylation followed by streptavidin-agarose affinity [15], and the crude exosomes consisted of the pellet of the cell supernatant centrifugation at 100,000xg (this fraction is enriched in exosomes and also contains other vesicles that are produced by the cells) [5,6]. MAL detected a major band at approximately 110 kDa in the crude exosomes, which was only faintly detected in the microsomal fraction or in the plasma membrane ( Figure 1A, indicated with an asterisk). SNA detected two bands at apparent molecular mass of 260 kDa and between 80 and 110 kDa, which were almost not detected in the microsomal fraction nor in the plasma membrane ( Figure  1A, indicated with asterisks). Digestion with sialidases from Vibrio cholerae, Artrobacter urefaciens (cleave α2,3/6-linked sialic acid) and Streptococcus pneumoniae (cleaves α2,3linked sialic acid) abolished MAL binding, whereas digestion with the former two but not the later abolished SNA binding thus corroborating the specificity of binding as previously reported [6]. Concanavalin A also showed a specific binding pattern for the crude exosomes. Incubation in the presence of 0.2 M methyl-α-D-mannopyranoside abolished detection corroborating the specificity of binding. These results showed that the glycosylation or glycoprotein profiles of crude exosomes were distinct from those of the microsomal fraction or plasma membrane.
We then aimed at identifying the MAL-binding glycoprotein that was enriched in exosomes in view of its abundance and specificity. With that purpose solubilized proteins from crude exosomes were fractionated by MAL affinity chromatography ( Figure 1B). Marked bands 1 to 21 ( Figure 1B) have been excised and analysed by peptide mass fingerprinting after trypsin hydrolysis. For comparison bands a to j (Figure 1B) from crude exosomes were also analysed by peptide mass fingerprinting ( Table 1). Identified proteins are shown in Table  1. Several glycoproteins have been identified in the MAL eluate: bands 1, 2, 4-8, 10, 12. Cytoplasmic proteins (11,(13)(14)(15)(16)(17)(18)(19)(20)(21) were also identified but they were due to non-specific binding to the resin since cytoplasmic glycoproteins do not contain sialic acid.
In the mass region of 100 kDa bands 4 to 8 and 10 ( Table 1) were identified as the galectin-3-binding protein (LGALS3BP).
LGALS3BP has a predicted mass of 65,331 based on the amino acid composition, but its detection around 100 kDa indicated that it is heavily glycosylated, and, therefore, the different bands 4 to 8 and 10 may correspond to distinct glycoforms, which is in agreement with the detection by MALlectin blotting as a broad band. Furthermore, the presence of glycans can impair the binding of the Coomassie dye to the protein thus explaining that a stronger detection was not observed [23].
Crude exosomes were further purified by sucrose gradient as previously described [5,6] and fractions were analysed by immunoblot for the conventional exosome marker Tsg101 (tumor susceptibility gene 101; protein involved in the formation of multivesicular endosomes) [24], CD9 [6] and LGALS3BP (Figure 2). Fractions 2-5 contained Tsg101, CD9 and also LGALS3BP (detected as a strong signal by immunoblot and MAL lectin blot). Since CD9 is also present at the plasma membrane and has recently been found in extracellular vesicles other than exosomes [24], biotinylated plasma membrane proteins were also analysed for comparison ( Figure  2A). As expected CD9 was detected at the plasma membrane contrary to Tsg101. Concerning LGALS3BP it was not detected at the plasma membrane which supported its specificity as an exosome marker.
Crude exosomes and purified exosomes were also visualized by electron microscopy. In agreement with the detection of biochemical markers, both preparations contained vesicles with exosome characteristics: cup-shaped vesicles up to approximately 100 nm (Figure 2 B,C, arrows), and smaller vesicles between approximately 30 and 50 nm (Figure 2 B,C, closed arrowheads). Larger vesicles or aggregates were also observed in crude exosomes and almost not detected in purified exosomes (Figure 2B, opened arrowhead).

N-glycosylation of glycoproteins from exosomes
N-Glycans have been released from glycoproteins of purified exosomes with peptide N-glycosidase F, fluorescently labeled with 2-aminobenzamide (2-AB) and analysed by NP-HPLC (Figure 3). Peaks corresponding to high mannose glycans were found in exosomes and identified by their retention times in comparison with the reference standards ( Figure S1, Table S1) and by their sensitivity toward mannosidase digestion ( Figure  3, second panel). Peaks corresponding to sialylated complex glycans were also found in exosomes, which corresponded to the peaks that disappeared after digestion with α2,3/6 specific sialidase (Figure 3, third panel). Ten desialylated complex glycans (shown in Figure 3, third panel) have been identified by comparison with the retention times of reference oligosaccharide standards (Figure S1; Table S1) and considering the profiles obtained after digestion with exoglycosidases (Figure 3). Complex glycans of the di-, tri-and tetraantennary type were found. Furthermore, diantennary glycans containing bisecting GlcNAc (peaks 3, 6) were also detected. Some of the structures were partially truncated (peaks 1, 2, 3), which could be due to the presence of glycosidases in the medium.
Desialylated N-glycans have also been analysed by MALDI-TOF MS (Figure 4). Based on the m/z values, the information from the NP-HPLC analysis/exoglycosidase digestion ( Figure  3) and the N-glycan biosynthetic pathway compatible glycan structures have been proposed for the major peaks ( Table 2). The exosomes contained complex glycans of the di-, tri-and tetraantennary type with or without proximal Fuc and high mannose glycans; structures lacking Gal residues have also been found ( Table 2). The presence of complex and high mannose glycans was also detected by MALDI-TOF MS in crude exosomes from another ovarian carcinoma cell line (OVM) (Figure S2).  (Figure S3). Fragments at m/z 1442.28 and 1460.39 derived from the parent ion at 2215.8 identified a triantennary complex glycan with proximal Fuc and bisecting GlcNAc (Figure S4). A mass signal at m/z 2215.8 by MALDI-TOF MS was also found in crude exosomes from the OVM ovarian carcinoma cell line ( Figure S2).

Discussion
Exosomes are secreted vesicles that are produced from the fusion of multivesicular bodies with the plasma membrane and present specific biochemical and molecular characteristics. In  Table 1) and total vesicles proteins (bands a to j were identified in Table 1 LGALS3BP as an exosome marker from ovarian carcinoma cells and we also characterized the N-glycans from exosomes. LGALS3BP is a secreted glycoprotein that has been found in the extracellular matrix and in body fluids, in exosomes from cancer cells, urinary and plasma exosomes [25] and in prostasomes, which are exosome-like vesicles secreted by the prostate [26]. It has been found in several cancers where it has a negative prognostic value, it mediates cell adhesion [27] and it has immunostimulatory properties [28]. Exosomes interact with target cells and, therefore, they are responsible for the transmission of pathogenic proteins among cells in neurodegenerative diseases and also for the activation of the immune system [1]. Several mechanisms underlie the exosome/target cell recognition that include ligand-receptor interactions, with subsequent fusion of vesicles with the plasma membrane or internalization of whole vesicles via endocytic pathways [1,6]. Several proteins have been shown to be involved in the interaction, for example, integrins (beta1 and beta2) from B cell exosomes mediate adhesion to collagen-I, fibronectin, and tumor necrosis factor-alpha-activated fibroblasts [29], and the intercellular adhesion molecule 1 from exosomes mediates interaction with the lymphocyte functionassociated antigen 1 underlying exosome capture in cells of the immune system [1]. To our knowledge LGALS3BP has not previously been described to be involved in exosome/target cell interaction, however, since it is known to bind several proteins that are found on the cell surface, such as, collagens IV, V, and VI, fibronectin and nidogen and galectin-3 [30] and integrin, beta1 [31], it is possible that due to its enrichment in exosomes it could play an important role in exosome/target cell interaction via components of the extracellular matrix with implications in exosome uptake. Further experiments are required to elucidate     Table 2.
doi: 10.1371/journal.pone.0078631.g004 LGALS3BP has seven potential N-glycosylation sites which are occupied for truncated forms expressed in HEK293 cells [30]. The extensive N-glycosylation explains the broad band that we have observed around 110 kDa. The strong binding by the lectin MAL shows that it contains sialic acid and indicates that most of the glycans are of the complex-type. Further studies are required to elucidate if certain glycoforms are specifically enriched in the exosomes.
Several other MAL-binding glycoproteins have also been identified in crude exosomes, such as laminin or cathepsin D, that had already been found by others in exosomes and are presented in the database Exocarta 2012 [25].
Concerning protein N-glycosylation in exosomes, the predominance of complex glycans has been found, with relatively high amounts of sialylation. This is in agreement with other reports where the enrichment of complex N-linked glycans into tumor microvesicles has been found [10]. However, exosomes also contained high mannose glycans, which is in agreement with the finding of this type of structures hexose.
These mass signals have been detected from independent preparations of purified exosomes (spectrum shown in Figure 4) and crude exosomes (data not shown). in urinary exovesicles [32] and in tumor microvesicles [10]. Initial results from our laboratory have indicated higher amounts of sialylated glycans by NP-HPLC in the exosomes relatively to plasma membrane or microsomal fraction (results not shown). Bisecting GlcNAc-containing N-glycans have been found in exosomes (Figure 3, Figure S3 and Figure S4). Accordingly, complex bisecting glycans have also been described in tissues of endometrioid ovarian cancer [33], and increased N-acetylglucosaminyltransferase III activity was found in human serum of hepatoma patients [34] and pancreatic carcinoma tissues [35]. However, in other types of cancer, for example, colorectal cancer, decreases in the levels of bisecting GlcNAc-containing structures have been observed [36]. Furthermore, the presence of bisecting GlcNAc has been associated with decreases in metastasis formation in several cancers [37]. In view of this, it will be necessary to further investigate the levels of bisecting GlcNAc in other ovarian cancer cell lines, in human tissues of different types and stages of ovarian cancer, and to study its specificity and functional role in the disease.
Exosomes have been used in nanoscale cancer vaccines for different types of cancer and although the results are promising, further studies need to be performed to engineer exosomes for cancer vaccine development [38]. Since glycosylation is a major hallmark of cancer where strong deregulation takes place one possible approach to improve the properties of exosomes as vaccines is to remodel their glycosylation, and in this context, knowing the detailed glycosylation properties of exosomes is of crucial importance. Furthermore, using LGALS3BP as a target to improve exosomes properties as vaccines also appears an appealing idea.

Supporting Information
Structures of 2-AB labeled reference oligosaccharide standards shown in Figure S1. (PDF)