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

• Published in: Scientific reports Vol. 9; no. 1; pp. 17387 - 16
• Main Authors: Zaborowski, Mikołaj Piotr, Cheah, Pike See, Zhang, Xuan, Bushko, Isabella, Lee, Kyungheon, Sammarco, Alessandro, Zappulli, Valentina, Maas, Sybren Lein Nikola, Allen, Ryan M., Rumde, Purva, György, Bence, Aufiero, Massimo, Schweiger, Markus W., Lai, Charles Pin- Kuang, Weissleder, Ralph, Lee, Hakho, Vickers, Kasey C., Tannous, Bakhos A., Breakefield, Xandra O.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 22.11.2019; Nature Publishing Group
• Subjects: 101/28; 13/1; 13/44; 631/1647/2230/1378; 631/80/313/2376; 64; 64/60; 82/16; 82/83; Animals; Cell interactions; Cell Line, Tumor; Cell signaling; Copepoda - enzymology; Copepoda - genetics; Copepoda - metabolism; Extracellular vesicles; Extracellular Vesicles - metabolism; Female; Gaussia; Humanities and Social Sciences; Humans; Luciferases - genetics; Luciferases - metabolism; Membrane proteins; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membranes; Membranes - metabolism; Mice; Mice, Nude; multidisciplinary; Proteins; Proteolysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Science; Science (multidisciplinary); Secretory Pathway - genetics; Tissue Distribution
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-53554-y

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.