title: A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol.
abstract: The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.
authors.affiliation: Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Zala Jenko
authors.initials: ZJ
authors.lastname: Praznikar
authors.forename: Lidija
authors.initials: L
authors.lastname: Kovacic
authors.forename: Edward G
authors.initials: EG
authors.lastname: Rowan
authors.forename: Rok
authors.initials: R
authors.lastname: Romih
authors.forename: Paola
authors.initials: P
authors.lastname: Rusmini
authors.forename: Angelo
authors.initials: A
authors.lastname: Poletti
authors.forename: Igor
authors.initials: I
authors.lastname: Krizaj
authors.forename: Joze
authors.initials: J
authors.lastname: Pungercar
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D009046:Motor Neurons / Q000166:cytology / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D054497:Phospholipases A2, Secretory / Q000235:genetics / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D021381:Protein Transport; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D014757:Viper Venoms / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D000199:Actins; D002147:Calmodulin; D014757:Viper Venoms; C068117:ammodytoxin A; D054497:Phospholipases A2, Secretory