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Different isolation approaches lead to diverse glycosylated extracellular vesicle populations.
Freitas D1,2,3,
Balmaña M1,2,
Poças J1,2,3,
Campos D1,2,
Osório H1,2,4,
Konstantinidi A5,
Vakhrushev SY5,
Magalhães A1,2,
Reis CA1,2,3,4.
- 1
- i3S-Institute for Research and Innovation in Health, University of Porto, Porto, Portugal.
- 2
- IPATIMUP -Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal.
- 3
- Instituto de Ciências Biomédicas Abel Salazar (ICBAS), University of Porto, Porto, Portugal.
- 4
- Faculty of Medicine of the University of Porto, Porto, Portugal.
- 5
- Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates.
KEYWORDS:
Extracellular vesicles; OptiPrep density gradient; glycosylation; isolation protocols; size exclusion chromatography; total exosome isolation; ultracentrifugation
Figure 1.
Schematic representation of the four methodologies applied for extracellular vesicles (EVs) isolation and further EV profiling and glycosylation characterization. EVs were isolated by ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) methodologies. ODG and SEC were applied after UC washing step (EVs*). TEI solution was applied directly to the conditioned medium (without previous UC).
J Extracell Vesicles. 2019;8(1):1621131.
Figure 2.
Representative example of the 12 fractions obtained by OptiPrepTM density gradient. Fractions were weight scaled for density evaluation and syntenin-1 expression was assessed. Fractions 6 and 7 showed a density of approximately 1.1 g/mL and the highest expression of syntenin-1 extracellular vesicle marker.
J Extracell Vesicles. 2019;8(1):1621131.
Figure 5.
Identification by MS of representative proteins from lysates of extracellular vesicles. Silver staining of extracellular vesicle lysates obtained by different isolation methodologies from MKN45 (M) human gastric cancer cell line and glycoengineered MKN45 (gM) cultured 48 h with 1640 RPMI (-FBS) or with 1640 RPMI supplemented with 10% FBS (+FBS). The total cell lysates are also shown. *These bands were identified with a high number of candidate peptides without reaching statistical significance.
J Extracell Vesicles. 2019;8(1):1621131.
Figure 6.
Protein profile of extracellular vesicles (EVs) isolated by differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) from MKN45 (M) human gastric cancer cell line and glycoengineered MKN45 (gM) cells. (a) Western blotting of HSP70, syntenin-1, CD9, CD63, Alix and CD81 extracellular vesicle markers and cytochrome C mitochondria marker performed on total cell lysates and EV lysates in non-FBS supplemented medium or (b) cells cultured with medium supplemented with 10% FBS. At least two independent experiments were conducted. (c) Western blotting of syntenin-1, CD9 and CD63 EV markers on total cell lysates and EV lysates in non-FBS supplemented medium. EV samples were isolated by UC or ODG followed by TEI.
J Extracell Vesicles. 2019;8(1):1621131.
Figure 7.
Glycan detection of extracellular vesicles obtained by differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) in the absence (-FBS) or presence (+FBS) of FBS in the culture medium. Western blotting of total cell lysates and extracellular vesicle lysates secreted by MKN45 (M) human gastric cancer cell line and glycoengineered MKN45 (gM) using (a) an anti-STn antibody (red dots indicate high molecular weight glycoproteins displaying STn) and the different lectins (b) E-PHA, (c) L-PHA and (d) AAL. Glycan epitopes recognized by the lectins and the antibody used are depicted (left panel). At least two independent experiments were conducted. The right panel shows the control condition performed with total cell lysates and the pellet obtained after UC of medium supplemented with 10% FBS and without cell contact. Abbreviations: STn: sialyl-Tn; AAL: Aleuria aurantia lectin; E-PHA: Phaseolus vulgaris erythroagglutinin; L-PHA: Phaseolus vulgaris leucoagglutinin.
J Extracell Vesicles. 2019;8(1):1621131.