Secretory phospholipases A2 (sPLA2) are a family of phospholipase A2 enzymes that catalyze the hydrolysis of the ester bond in glycerophospholipids at the sn-2 position, releasing free fatty acids and lysophospholipids. However, sPLA2s can also act as ligands for diverse protein receptors, making their (patho)physiology complex and, despite intensive research, often unexplained at the molecular level. Among the best studied is the molecular mechanism of action of β-neurotoxic sPLA2s from snake venoms (β-ntx). β-ntx damage nerve endings, resulting in the blockade of neuromuscular transmission and subsequent flaccid paralysis. In this process, interaction of β-ntx with mitochondria plays a key role. Mammalian group IIA sPLA2 (GIIA) can also have toxic effects on nerve cells under pathological conditions, e.g. in some neurodegenerative diseases. Since mammalian GIIA and viperid β-ntx are orthologous molecules, the observed effects could have the same molecular basis. To gain a better insight into the (patho)physiological action of mammalian and β-neurotoxic GIIA sPLA2s at the molecular level, we investigated the processes triggered by ammodytoxin (Atx) and its rat ortholog (rGIIA) in the mitochondria of nerve cells and investigated their uptake and localization in these cells. We prepared recombinant Atx and rGIIA and their enzymatically inactive mutants, Atx(D49S) and rGIIA(D49S). We showed that all four products interact with cytochrome c oxidase subunit II (CCOX-II). rGIIA and rGIIA(D49S) bound to this essential complex of the respiratory chain with approximately 100-fold lower affinity than Atx. Nevertheless, both rGIIA molecules strongly inhibited CCOX enzyme activity in isolated mitochondria from PC12 cells. rGIIA also bound to a mitochondrial protein with an apparent molecular mass of 20 kDa, however, the identity of this receptor remains unknown. With transmission electron and confocal microscopy, we showed that Atx and rGIIA internalize into PC12 cells and colocalize with mitochondria independently of their phospholipase activity. This suggests that both sPLA2s use the same pathway for cellular uptake. Atx(D49S) and rGIIA(D49S) also had a similar inhibitory effect on mitochondrial membrane potential and CCOX activitiy in PC12 cells and rat brain tissue sections, respectively. The results therefore show that β-neurotoxic GIIA from snake venoms and mammalian GIIA share a similar molecular background in their effects on neuronal mitochondria, suggesting a possible mechanism by which mammalian GIIA is involved in Alzheimer’s disease. In an effort to develop new procedures for diagnosis, prevention and treatment of Alzheimer's and related neurodegenerative diseases, the results of this work will be a good starting point for testing GIIA as a new early marker and potential therapeutic target in these diseases.
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