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

Abstract

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.

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

“…Taken together, our glycomics study demonstrated the prevalence of complex N -glycans in all particle subsets with relatively high levels of sialylation, consistent with previous findings of complex N -glycans and sialoglycoproteins in tumor microvesicles/exosomes 25-27 . Furthermore, our study revealed differences in N- glycan composition and structures among exomeres, Exo-S, and Exo-L.…”

“…Similar to the expression in metastatic tumor cells, exosome subsets were enriched with sialylated glycoproteins, supporting the role of these structures in exosome-mediated cellular recognition. One predominant sialoglycoprotein previously identified in exosomes 25, 35 , the galectin-3-binding protein (LGALS3BP), a modulator of cell communication and immune responses 36, 37 , was highly enriched in exomeres. This ligand could mediate the specific interaction of exomeres with target cells through proteins, such as collagens, fibronectin, nidogen, galectin-3 and integrin beta-1 38,39 .…”

Post-translational add-ons mark the path in exosomal protein sorting

“…Of these, 21% are transmembrane proteins, underscoring that deubiquitination is not mandatory for the incorporation of protein cargo into MVBs and exosomes [84]. Although, ubiquitination-specific motifs remain unidentified, this PTM has preference for basic * More proteins idenƟfied in a high-throughput screening approach, proteomics [74,84,101,108,120]. # Mycobacterial proteins detected in exosomes from human and mouse cell lines.…”

“…# Mycobacterial proteins detected in exosomes from human and mouse cell lines. $ More glycosylated proteins idenƟfied by lecƟn bloƫng, NP-HPLC analysis and mass spectrometry [120].…”

“…Notably, EVs contain mainly mannose or sialic acid enriched-glycans and N-linked glycoproteins. In particular, exosomes contain large amounts of the sialoglycoprotein galectin-3-binding protein (LGALS3BP) [119][120][121][122] (Table 2).…”