Amniotic fluid- derived extracellular vesicles, an unconventional localization for enzymes involved in ATP turnover.

Glycolytic enzymes are commonly detected in proteomic profiling of exosomes of different sources representing frequently the most identified proteins. However, the metabolic role these enzymes in exosomes and in general in extracellular vesicles (EVs) is not yet known. Several open questions remain such as: (i) can EVs bring glycolysis and produce lactate and ATP? (ii) which would be the biological function of ATP produced by EVs? Here, we demonstrated, that amniotic fluid-derived extracellular vesicles (AF-EVs) carry out glycolytic enzymes, glucose transporters and other enzymes involved in ATP turnover. Moreover, AF-EVs can produce ATP not only through glycolysis but also through the adenylate kinase activity. Notably, we found the uptake of AF-EVs by human amniotic stem cells (HASCs), was dependent upon an ongoing glycolytic flux, involving extracellular ATP production by EVs. We have also shown that ATP produced by EVs may be involved in cell reprogramming of THP-1 monocytes. Indeed, in these cells we demonstrated that AF-EVs, highly roducing ATP, induced a significant reduction of cell viability and this effect was associated with pyroapototic-morphological changes, suggesting an immune-odulatory role of AF-EVs producing extracellular adenosine. Accordingly, we found that AF-EVs possess also the enzymes required to convert ATP into adenosine, such as CD39 (ecto- nucleoside triphosphate diphosphohydrolase 1) and CD73 (ecto-5’nucleotidase). In agreement with this hypothesis, the adenosine receptor agonist NECA (1-(6-Amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide) mirrored the effect induced by AF-EVs in THP-1 cells, whereas the adenosine receptor antagonist Caffeine reverted the AF-EVs-induced effects. In conclusion, in this work we demonstrated for the first time that AF-EVs bring glycolytic enzymes that produce ATP, required for AF-EVs uptake by target cells. Moreover, we also showed that AF-EVs express ecto-nucletide enzymes, involved in further ATP metabolism to adenosine promoting immune regulatory functions in monocytes.