Assessing the role of surface glycans of extracellular vesicles on cellular uptake

Charles Williams,Esperanza González,Jorge Gamiz,Félix Royo,Raquel Pazos,Niels-Christian Reichardt,Juan M Falcón-Pérez,Aitor Martinez,Meritxell Roura-Ferrer

doi:10.1038/s41598-019-48499-1

Abstract

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

Highlights

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts
Intact EVs purified from AML12 and MLP29 cell cultures were fluorescently labelled with Alexa Fluor 647 and incubated with microarrays comprising 47 lectins, before washing and fluorescence scanning (Fig. 1)
Similar lectin binding profiles were observed for both types of vesicles, with high fluorescent signals observed for N-acetylglucosamine binding lectins wheat germ agglutinin (WGA), Phytolacca americana (PWA) and Pseudomonas aeruginosa PA-I (PAL), amongst several other lectins such as Marasmium oreades agglutinin (MOA) and Euonymus europaeus (EEA)

Summary

Introduction

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. There is ambiguity around the fundamental mechanisms by which these effects are transduced, in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Uptake of exosomes was depressed through interference of sialic acid-binding immunoglobulin-type lectin (siglec) receptors. This effect was observed in vitro through antibody and free monosaccharide inhibition of Siglecs in HeLa cell culture[21] and in vivo, with murine gene knockouts of Siglec-122. Desialylation of EVs has been shown to alter their biodistribution in vivo[23]

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