A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol.
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.
Praznikar
Zala Jenko
ZJ
Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Ljubljana, Slovenia.
Kovacic
Lidija
L
Rowan
Edward G
EG
Romih
Rok
R
Rusmini
Paola
P
Poletti
Angelo
A
Krizaj
Igor
I
Pungercar
Joze
J
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*
D048948:14-3-3 Proteins; D000199:Actins; D002147:Calmodulin; D014757:Viper Venoms; C068117:ammodytoxin A; D054497:Phospholipases A2, Secretory