Restoration of enzymatic activity in a Ser-49 phospholipase A2 homologue decreases its Ca(2+)-independent membrane-damaging activity and increases its toxicity.
Ammodytin L (AtnL) is a Ser-49 secretory phospholipase A2 (sPLA2) homologue with myotoxic activity. By analogy to the Lys-49 sPLA2 myotoxins, AtnL has been predicted to be enzymatically inactive due to the absence of the conserved Asp-49 that participates in coordination of the Ca2+ cofactor. By substituting Ser-49 and three other residues in the Ca2+-binding loop of AtnL, we obtained the first two enzymatically active mutants of Lys-49/Ser-49 sPLA2 homologues. The mutants LW and LV, which differed only by the presence of Trp and Val at position 31, respectively, efficiently hydrolyzed phospholipid vesicles, while recombinant AtnL displayed no activity. In contrast to AtnL but similarly to ammodytoxin A (AtxA), a homologous neurotoxic sPLA2, both mutants exhibited catalysis-dependent membrane-damaging ability, involving vesicle contents leakage and fusion. However, LW and LV also exhibited the potent, Ca2+-independent disruption of vesicle integrity characteristic of AtnL, but not of AtxA, in which leakage of the contents is not associated with membrane fusion. Although LV and, especially, LW have the advantage over AtnL of being able to act in both Ca2+-independent and Ca2+-dependent modes, and display higher cytotoxicity and higher lethal potency, they have a lower Ca2+-independent membrane-damaging potency and display reduced specificity in targeting muscle fibers in vitro. Our results indicate that, in evolution, Lys-49 and Ser-49 sPLA2 myotoxins have lost their Ca2+-binding ability and enzymatic activity through subtle changes in the Ca2+-binding network without affecting the rest of the catalytic machinery, thereby optimizing their Ca2+-independent membrane-damaging ability and myotoxic activity.
Petan
Toni
T
Department of Molecular and Biomedical Sciences, JoZef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Krizaj
Igor
I
Pungercar
Joze
J
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects*; D002478:Cells, Cultured; D003603:Cytotoxins / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins / Q000494:pharmacology; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism / Q000494:pharmacology; D054497:Phospholipases A2, Secretory / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D010743:Phospholipids / Q000737:chemistry; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D053835:Unilamellar Liposomes / Q000737:chemistry; D014757:Viper Venoms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D003603:Cytotoxins; D005452:Fluoresceins; D050505:Mutant Proteins; D010743:Phospholipids; D053835:Unilamellar Liposomes; D014757:Viper Venoms; C065419:ammodytin L; D012694:Serine; D054497:Phospholipases A2, Secretory; D002118:Calcium; C007740:fluorexon