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

Abstract

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.

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer’s Disease

“…Like Atx, a snake venom orthologue of rGIIA, rGIIA was also able to reach the mitochondria of the PC12 cells from the media ( Supplementary Figure S1 ). Just recently, it was reported that Atx was able to enter the mitochondria of the PC12 cells independently of its enzyme activity [ 14 ]; so, we also tested such an ability for the enzymatically inactive mutant of rGIIA, rGIIA(D49S). The mutant was fluorescently labeled with 546 Alexa and added to the PC12 cells in a culture.…”

“…This activity of GIIA is obviously independent of its phospholipase activity, as is its cell internalization. Indeed, rGIIA(D49S) added to the medium containing PC12 cells passed through the plasma membrane and colocalized with mitochondria like Atx(D49S) [ 14 ]. The effect of rGIIA(D49S) on the cells in the culture was indicated by a slightly reduced TMRM fluorescence intensity, indicating a reduction in the MMP.…”

“…rGIIA and rGIIA(D49S) were expressed in the form of inclusion bodies in E. coli BL21 (DE3). The in vitro refolding of the inclusion-body proteins proceeded as described in [ 14 ].…”

“…Mitochondria were prepared from PC12 cells, as described in Ivanušec et al [ 14 ], and the CCOX activity was measured using the Cytochrome c Oxidase Assay Kit (Sigma-Aldrich, St. Louis, MO, USA), as described previously [ 13 ]. Briefly, the isolated mitochondria (12 µg/mL total protein) were incubated for 30 min at room temperature in the presence of either 1 µM rGIIA or 1 µM rGIIA(D49S).…”

“…Consequently, it inhibited CCOX activity. As has just been shown, Atx is transported from the extracellular space to the neuronal mitochondria via a specific pathway, without the need to be phospholipolytically active [ 14 ].…”