A Secreted Phospholipase A₂ Binds to Calmodulin at Sub-micromolar Concentrations of Calcium

Uroš Logonder,¹ Jernej Jorgačevski,¹ Gregor Anderluh,² Uroš Petrovič,¹ Igor Poberaj³ and Igor Križaj^1,*

¹Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia
²Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia
³Department of Physics, Faculty of Mathematics and Physics, University of Ljubljana, Jadranska cesta 19, SI-1000 Ljubljana, Slovenia
* Corresponding author: E-mail: igor.krizaj@ijs.si,
Tel: +386 1 477 36 26, Fax: +386 1 477 39 84

Abstract
To determine the possibility that calmodulin, a cytosolic regulatory protein, and ammodytoxin, a neurotoxic secreted phospholipase A₂, interact in vivo, we studied the dependence of their association on Ca²⁺. The interaction between the two proteins was positively dependent on Ca²⁺, and greatest at millimolar concentrations of this ion. Importantly, they nteracted already in the presence of sub-micromolar concentrations of Ca²⁺, as demonstrated by affinity labelling, laser tweezers and surface plasmon resonance. Tight binding of the secreted phospholipase A₂ to calmodulin is therefore expected on depolarization of the axolemma, when the concentration of free Ca²⁺ at active zones rises to 100 µM. These results strengthen the proposal that binding of ammodytoxin to calmodulin is a step in the process of presynaptic toxicity of this and related phospholipases A₂. Moreover, they suggest that some other (patho)physiological effects induced by endogenous secreted phospholipases A₂ could be also due to their interaction with calmodulin in the cytosol of cells.

Keywords: Secreted phospholipase A₂, Ammodytoxin, Calmodulin, Neurotoxicity, Ca²⁺-dependence, Cytosol