Functional Properties of Circulating Exosomes Mediated by Surface-Attached Plasma Proteins

Abstract

Background: Exosomes and other types of extracellular vesicles present an important component of circulating plasma. Exosomes released by endothelial and blood cells account for majority of plasma exosomal population; exosomes secreted by other cells might cross tissue-plasma barrier and reach circulating plasma as well. Definitely, exosomes of different cellular origins are different by content and function. However, exosomal surface membrane interacts with plasma components. This interaction may alter composition of exosomal surface and hence, provide these vesicles with new functional properties. This study was aimed to estimate composition and possible functional role of proteins attached on the surface of plasma exosomes. Methods: Here, extracellular vesicles from human plasma were isolated by ultracentrifugation and treated by trypsin. Trypsinized and native exosomes were analyzed by nanoparticle tracking analysis, Western blotting and quantitative high-resolution mass spectrometry. Results: Surface-attached proteins were removed from exosomes isolated from plasma of healthy donors by incubation with serine protease (trypsin). Treatment did not impact exosomes integrity while slightly reduced hydrodynamic radius. Mass spectrometry revealed 259 exosomal proteins; among them 79 proteins were completely removed and more than half of the proteins were partially removed by trypsinization. Gene ontology functional annotation revealed mostly extracellular locations of proteins cleaved from a surface of the plasma exosomes. Moreover, proteins cleaved from the exosome surface are supposed to be implicated into integrin-linked kinase (ILK), focal adhesion kinase (FAK) and other pathways connecting cell surface with intracellular signaling cascades. Conclusion: Taken together, our results demonstrate that a surface of circulating exosomes is decorated by plasma proteins, and these proteins can mask tissue-specific characteristic of the exosomal surface membrane and provide exosomes with new and uniform properties.

Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo—Implications for biomarker discovery

“…This study reported 56 common proteins with our proteome from dUC and DG samples, including complement proteins, immunoglobulins, apolipoproteins and keratins. Similar results have also been reported in studies using dUC [31][32][33][34] or SEC as isolation method [35][36][37]. In the study of Karimi et al it was shown that by combining dUC, DG and SEC, it is possible to avoid lipoprotein and soluble protein contamination and to isolate highly purified plasma EVs for proteomic analysis [36].…”

“…Study samples were collected twice in Helsinki (set I and II) and once in Budapest (set III), the latter to perform fluorescence size exclusion chromatography (Flu-SEC). The average age of the donors in set I was 45 years (range 27-55), 32 years (range 18-51 years) in set II, and 30 years (range [25][26][27][28][29][30][31][32][33][34][35][36] in set III. Majority of the volunteers were females (80%, 70%, and 67%).…”

Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid

“…Taking into account the obtained data on the concentration of vesicles in CSF, as well as our own experience in visualization of EVs from different biofluids by cryo-EM [20][21][22] we pooled all particle samples (from about 30 mL of CSF for PD patients and 50 mL for the comparison group) for re-ultracentrifugation to maximize the number of EVs in the final pellet. NTA of the sample of vesicles isolated from pooled CSF of PD patients showed that the particles generally varied in size from 26 to 305 nm and that the vesicular size mode, D90 and concentration were 69 nm, 125 nm and 0.6x10 12 particles/ml, respectively ( Fig 1B).…”

Evaluation of immune and chemical precipitation methods for plasma exosome isolation

“…Most exosomes contain proteins that are common among all exosomes regardless of the types of cells which secrete them [ 33 , 34 ]. These characteristic proteins therefore may serve as convenient biomarkers for the isolation and quantification of exosomes.…”