Abstract
Cells release vesicles to the extracellular environment with characteristic nucleic acid, protein, lipid, and glycan composition. Here we have isolated and characterized extracellular vesicles (EVs) and total cell membranes (MBs) from ovarian carcinoma OVMz cells. EVs were enriched in specific markers, including Tsg101, CD63, CD9, annexin-I, and MBs contained markers of cellular membrane compartments, including calnexin, GRASP65, GS28, LAMP-1, and L1CAM. The glycoprotein galectin-3 binding protein (LGALS3BP) was strongly enriched in EVs and it contained sialylated complex N-glycans. Lectin blotting with a panel of lectins showed that EVs had specific glycosignatures relative to MBs. Furthermore, the presence of glycoproteins bearing complex N-glycans with α2,3-linked sialic acid, fucose, bisecting-GlcNAc and LacdiNAc structures, and O-glycans with the T-antigen were detected. The inhibition of N-glycosylation processing from high mannose to complex glycans using kifunensine caused changes in the composition of EVs and induced a decrease of several glycoproteins. In conclusion, the results showed that glycosignatures of EVs were specific and altered glycosylation within the cell affected the composition and/or dynamics of EVs release. Furthermore, the identified glycosignatures of EVs could provide novel biomarkers for ovarian cancer.
Highlights
Extracellular vesicles (EVs) are produced by virtually all cells and a large body of evidence for their biological relevance has been obtained for immune, tumor or neural cells
In the present work we showed that extracellular vesicles (EVs) from ovarian carcinoma cells have specific glycosignatures using lectin blotting, which may constitute potential biomarkers
EVs were isolated from confluent monolayers of OVMz ovarian carcinoma cells grown in serum depleted medium for 48 h
Summary
Extracellular vesicles (EVs) are produced by virtually all cells and a large body of evidence for their biological relevance has been obtained for immune, tumor or neural cells. The EVs may have different cellular origins, either from the multivesicular endosomes (designated as exosomes) or from budding off the plasma membrane (commonly known as microvesicles) [1]. EVs include apoptotic vesicles that usually have larger diameters, ranging from 50–5000 nm [1,2]. In view of the overlapping of several biochemical and biophysical characteristics, the discrimination of the different vesicle types is a complex topic due to limitations of the techniques used for their purification. It has been suggested that from endosomal origin there are different types of exosomes. The methodology for isolation of pure vesicle fractions is a challenging topic and, currently, several techniques are used, including ultracentrifugation, ultrafiltration, gel exclusion chromatography, immunoaffinity isolation [3], and lectin affinity separation [4]. Because of the different methodologies used for EVs isolation and characterization, a consensus article aiming at standardization of sample collection, isolation, and analysis methods for EVs research has recently been published [5]
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