The Phospholipase Activity of Ammodytoxin, a Prototype Snake Venom β-Neurotoxin, Is Not Obligatory for Cell Internalisation and Translocation to Mitochondria

β-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype β-neurotoxin from the venom of the nos...

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Bibliographic Details
Published in:Toxins 2022-05, Vol.14 (6), p.375
Main Authors: Ivanušec, Adrijan, Šribar, Jernej, Veranič, Peter, Križaj, Igor
Format: Article
Language:English
Subjects:
ammodytoxin
Antibodies
Cytosol
Endoplasmic reticulum
Enzymatic activity
Fluorescence microscopy
Localization
Microscopy
Mitochondria
Morphology
Motor neurons
Nanoparticles
Nerve endings
Neuromuscular junctions
Neurons
Neurotoxicity
Neurotoxins
Pheochromocytoma cells
Phospholipase
Phospholipase A2
Proteins
Prototypes
secreted phospholipase A2
snake venom
Synapses
Toxins
Translocation
Transmission electron microscopy
Venom
Vipera ammodytes ammodytes
β-neurotoxicity
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Summary:β-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype β-neurotoxin from the venom of the nose-horned viper (Vipera ammodytes ammodytes). Here, we studied the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement using transmission electron microscopy (TEM). We prepared a recombinant, enzymatically inactive mutant of Atx, Atx(D49S), labelled with gold nanoparticles (GNP), and incubated this with PC12 cells, to analyse its localisation by TEM. Atx(D49S)-GNP internalised into the cells. Inside the cells, Atx(D49S)-GNP was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Co-localization of fluorescently labelled Atx(D49S) with mitochondria in PC12 cells by confocal fluorescence microscopy confirmed the reliability of results generated using Atx(D49S)-GNP and TEM and allowed us to conclude that the phospholipase activity of Atx is not obligatory for its cell internalisation and translocation into the mitochondrial intermembrane space and matrix.
ISSN:2072-6651
2072-6651
DOI:10.3390/toxins14060375