Cell surface nucleolin interacts with and internalizes Bothrops asper Lys49 phospholipase A2 and mediates its toxic activity

Maria Lina Massimino,Fiorella Tonello,Bruno Lomonte,Barbara Spolaore,Giorgio Arrigoni,Cinzia Franchin,Morena Simonato,Laura Monturiol-Gross,Julián Fernández,Oriano Marin

doi:10.1038/s41598-018-28846-4

Maria Lina Massimino, Fiorella Tonello + Show 8 more

Open Access

https://doi.org/10.1038/s41598-018-28846-4

Copy DOI

Abstract

Phospholipases A2 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 PLA2s are not directly connected. In addition, viperid venoms contain PLA2-like proteins, which are very toxic even if they lack catalytic activity due to a critical mutation in position 49. In this work, the PLA2-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 °C, on cell membrane where they form Congo-red sensitive assemblies, while at 37 °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.

Highlights

Secreted PLA2s are proteins of about 14 kDa with a conserved tridimensional structure composed of three main alpha helices, a beta sheet and seven disulphide bonds
The currently held view is that myotoxin II (Mt-II) and other PLA2-like myotoxins exert their toxic activity by affecting the plasma membrane integrity by interaction with membrane lipids, with consequent rapid influx of calcium ions that eventually triggers a series of degenerative events[11]
Mt-II-TAMRA added to culture of primary mouse myotubes and of RAW264.7 macrophages is internalized in both kinds of cell (Fig. 1)

Summary

Introduction

Secreted PLA2s (sPLA2s) are proteins of about 14 kDa with a conserved tridimensional structure composed of three main alpha helices, a beta sheet and seven disulphide bonds They have been isolated for the first time from cobra venom and successively from mammalian pancreas, but they are present in about all mammalian tissues. The first mammalian sPLA2 receptor, PLA2R1, was identified by cross-linking experiments involving OS2, a PLA2 from the snake Oxyuranus scutellatus that displays both neurotoxic and local myotoxic activities[3]. This is of high relevance, in the light of the emerging involvement of mammalian sPLA2s in many human disorders[4,5,6]. The triggered signal could lead to toxin internalization, and the Mt-II cell entry could be a necessary step for its toxic activity

Methods

Results

Conclusion