Phospholipases A(2) are a major component of snake venoms. Some of them cause severe muscle necrosis through an unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA(2)s are not directly connected. In addition, viperid venoms contain PLA(2)-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA(2)-like Bothrops asper myotoxin-II, conjugated with the fluorophore TAMRA, was found to be internalized in mouse myotubes, and in RAW264.7 cells. Through experiments of protein fishing and mass spectrometry analysis, using biotinylated Mt-II as bait, we found fifteen proteins interacting with the toxin and among them nucleolin, a nucleolar protein present also on cell surface. By means of confocal microscopy, Mt-II and nucleolin were shown to colocalise, at 4 degrees C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 degrees C, 20 minutes after the intoxication, they colocalise in intracellular spots going from plasmatic membrane to paranuclear and nuclear area. Finally, nucleolin antagonists were found to inhibit the Mt-II internalization and toxic activity and were used to identify the nucleolin regions involved in the interaction with the toxin.

Cell surface nucleolin interacts with and internalizes Bothrops asper Lys49 phospholipase A2and mediates its toxic activity

Massimino, Maria Lina;Spolaore, Barbara;Franchin, Cinzia;Arrigoni, Giorgio;Marin, Oriano;Tonello, Fiorella
2018

Abstract

Phospholipases A(2) are a major component of snake venoms. Some of them cause severe muscle necrosis through an unknown mechanism. Phospholipid hydrolysis is a possible explanation of their toxic action, but catalytic and toxic properties of PLA(2)s are not directly connected. In addition, viperid venoms contain PLA(2)-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA(2)-like Bothrops asper myotoxin-II, conjugated with the fluorophore TAMRA, was found to be internalized in mouse myotubes, and in RAW264.7 cells. Through experiments of protein fishing and mass spectrometry analysis, using biotinylated Mt-II as bait, we found fifteen proteins interacting with the toxin and among them nucleolin, a nucleolar protein present also on cell surface. By means of confocal microscopy, Mt-II and nucleolin were shown to colocalise, at 4 degrees C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 degrees C, 20 minutes after the intoxication, they colocalise in intracellular spots going from plasmatic membrane to paranuclear and nuclear area. Finally, nucleolin antagonists were found to inhibit the Mt-II internalization and toxic activity and were used to identify the nucleolin regions involved in the interaction with the toxin.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3279146
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