Abstract
Extracellular vesicles (EVs) are membranous structures containing bioactive molecules, secreted by most cells into the extracellular environment. EVs are classified by their biogenesis mechanisms into two major subtypes: ectosomes (enriched in large EVs; lEVs), budding directly from the plasma membrane, which is common in both prokaryotes and eukaryotes, and exosomes (enriched in small EVs; sEVs) generated through the multivesicular bodies via the endomembrane system, which is unique to eukaryotes. Even though recent proteomic analyses have identified key proteins associated with EV subtypes, there has been no systematic analysis, thus far, to support the general validity and utility of current EV subtype separation methods, still largely dependent on physical properties, such as vesicular size and sedimentation. Here, we classified human EV proteomic datasets into two main categories based on distinct centrifugation protocols commonly used for isolating sEV or lEV fractions. We found characteristic, evolutionarily conserved profiles of sEV and lEV proteins linked to their respective biogenetic origins. This may suggest that the evolutionary trajectory of vesicular proteins may result in a membership bias toward specific EV subtypes. Protein–protein interaction (PPI) network analysis showed that vesicular proteins formed distinct clusters with proteins in the same EV fraction, providing evidence for the existence of EV subtype-specific protein recruiters. Moreover, we identified functional modules enriched in each fraction, including multivesicular body sorting for sEV, and mitochondria cellular respiration for lEV proteins. Our analysis successfully captured novel features of EVs embedded in heterogeneous proteomics studies and suggests specific protein markers and signatures to be used as quality controllers in the isolation procedure for subtype-enriched EV fractions.
Highlights
Extracellular vesicles (EVs) are membrane-bound particles that are secreted by cells across the evolutionary spectrum (Gill et al, 2019)
Ectosome-enriched like viral protein processing (lEV) fraction is often sedimented under 10,000 × g centrifugal force and exosome-enriched sEVs require over 100,000 × g preceded by pre-clearance of lEVs (Greening et al, 2015; Jeppesen et al, 2019)
We defined “Identification frequency in sEV datasets” and “Identification frequency in lEV datasets” of every vesicular protein identified in the integrated 254 human datasets in which the number of datasets that identified the protein divided by the whole number of sEV or lEV datasets, respectively
Summary
Extracellular vesicles (EVs) are membrane-bound particles that are secreted by cells across the evolutionary spectrum (Gill et al, 2019). There are two major subtypes of EVs based on their biogenesis and size (Cocucci and Meldolesi, 2015): (i) Ectosomes (100–1000 nm in diameter) bud from the cellular surface and are frequently referred to as microvesicles in eukaryotes, or as outer membrane vesicles in prokaryotes and (ii) exosomes (30–100 nm) arise within endosome-related intraluminal vesicles, which are released from cells upon recruitment of multivesicular bodies to the inner layer of the plasma membrane (Colombo et al, 2014; van Niel et al, 2018). The impact of evolutionary characteristics of biogenesis mechanisms on the vesicular cargo composition has not been explored
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