Ammodytoxins, potent presynaptic neurotoxins, are also highly efficient phospholipase A2 enzymes.
The enzymatic activity of ammodytoxins (Atxs), secreted phospholipases A(2) (sPLA(2)s) in snake venom, is essential for expression of their presynaptic neurotoxicity, but its exact role in the process is unknown. We have analyzed in detail the enzymatic properties of Atxs, their mutants, and homologues. The apparent rates of phospholipid hydrolysis by the sPLA(2)s tested vary by up to 4 orders of magnitude, and all enzymes display a strong preference for vesicles containing anionic phospholipids, phosphatidylglycerol or phosphatidylserine (PS), over those containing zwitterionic phosphatidylcholine (PC). Nevertheless, Atxs are quite efficient in hydrolyzing pure PC vesicles as well as PC-rich plasma membranes of intact HEK293 cells. The presence of anionic phospholipids in PC vesicles dramatically increases the interfacial binding affinity and catalytic activity of Atxs, but not of their nontoxic homologue ammodytin I(2), that displays unusually low binding affinity and enzymatic activity on PS-containing vesicles and HEK293 plasma membranes. Aromatic and hydrophobic residues on the interfacial binding surface of Atxs are important for productive binding to both zwitterionic and anionic vesicles, while basic and polar residues have a negative impact on binding to zwitterionic vesicles. When tightly bound to the membrane interface, Atxs can reach full enzymatic activity at low micromolar concentrations of Ca(2+). Although Atxs have evolved to function as potent neurotoxins that specifically target presynaptic nerve terminals, they display a high degree of phospholipolytic efficiency on various phospholipid membranes.
Petan
Toni
T
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Krizaj
Igor
I
Gelb
Michael H
MH
Pungercar
Joze
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D004791:Enzyme Inhibitors; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014757:Viper Venoms / Q000737:chemistry* / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D008081:Liposomes; D009498:Neurotoxins; D011994:Recombinant Proteins; D014757:Viper Venoms; D010741:Phospholipases A; D054467:Phospholipases A2