Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles

Mikołaj Piotr Zaborowski,Charles Pin- Kuang Lai,Ryan M Allen,Isabella Bushko,Ralph Weissleder,Alessandro Sammarco,Markus W Schweiger,Massimo Aufiero,Sybren Lein Nikola Maas,Hakho Lee,Xuan Zhang,Pike See Cheah,Valentina Zappulli,Kyungheon Lee,Xandra O Breakefield,Purva Rumde,Bence György,Bakhos A Tannous,Kasey C Vickers

doi:10.1038/s41598-019-53554-y

Abstract

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

Highlights

Extracellular vesicles (EVs) are membrane-encapsulated vesicles released by cells into the extracellular space[1]
To determine if membrane-bound version of Gaussia luciferase (mbGluc) is bound to non-EV material in the extracellular space and biofluids, we first evaluated mbGluc activity in conditioned medium collected from cancer cells without any EV isolation
Conditioned media was fractionated by sucrose/iodixanol gradients and mbGluc abundance was detected in fractions corresponding to EVs, and was readily detected in the very low-density fractions, which are attributed to free proteins

Summary

Introduction

Extracellular vesicles (EVs) are membrane-encapsulated vesicles released by cells into the extracellular space[1]. We previously demonstrated that a membrane-bound version of Gaussia luciferase (mbGluc), known as GlucB in previous reports, can be incorporated into the membranes of cells and EVs5,6 In these studies, EVs isolated by high-speed ultracentrifugation (UC) from cultured cells labeled with mbGluc were intravenously (i.v.) - injected into mice to monitor their biodistribution[6]. EVs isolated by high-speed ultracentrifugation (UC) from cultured cells labeled with mbGluc were intravenously (i.v.) - injected into mice to monitor their biodistribution[6] It is likely, that some methods of EV isolation, e.g. UC can Gynecologic Oncology, Division of Gynecologic Oncology, Poznan University of Medical Sciences, 60-535, Poznań, Poland. We used this knowledge to develop a novel assay to quantitatively track shedding of membrane proteins from the surface EVs both in culture and in vivo

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