The cationic cluster of group IVA phospholipase A2 (Lys488/Lys541/Lys543/Lys544) is involved in translocation of the enzyme to phagosomes in human macrophages

Javier Casas,Martín Valdearcos,José Pindado,Jesús Balsinde,María A Balboa

doi:10.1194/jlr.m001461

Abstract

Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) plays a role in the microbicidal machinery of immune cells by translocating to phagosomes to initiate the production of antimicrobial eicosanoids. In this work, we have studied the involvement of the cationic cluster of cPLA(2)alpha (Lys(488)/Lys(541)/Lys(543)/Lys(544)) in the translocation of the enzyme to the phagosomal cup in human macrophages responding to opsonized zymosan. Phagocytosis was accompanied by an increased mobilization of free arachidonic acid, which was strongly inhibited by pyrrophenone. In transfected cells, a catalytically active enhanced green fluorescent protein-cPLA(2)alpha translocated to the phagocytic cup, which was corroborated by frustrated phagocytosis experiments using immunoglobulin G-coated plates. However, a cPLA(2)alpha mutant in the polybasic cluster that cannot bind the anionic phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP(2)) did not translocate to the phagocytic cup. Moreover, an enhanced yellow fluorescent protein (EYFP)-cPLA(2)alpha and an enhanced cyan fluorescent protein-pleckstrin homology (PH) domain of the phospholipase Cdelta1 (PLCdelta(1)) construct that specifically recognizes endogenous PIP(2) in the cells both localized at the same sites on the phagosome. High cellular expression of the PH domain inhibited EYFP-cPLA(2)alpha translocation. On the other hand, group V-secreted phospholipase A(2) and group VIA calcium-independent phospholipase A(2) were also studied, but the results indicated that neither of these translocated to the phagosome. Collectively, these data indicate that the polybasic cluster of cPLA(2)alpha (Lys(488)/Lys(541)/Lys(543)/Lys(544)) regulates the subcellular localization of the enzyme in intact cells under physiologically relevant conditions.

Highlights

Using human macrophages responding to opsonized zymosan, we demonstrate in this work that translocation of cPLA2␣ to the phagocytic cup depends on an intact cationic cluster Lys488/Lys541/Lys543/ Lys544) in the catalytic domain of the enzyme
Experiments were performed next to rule out the possibility that the increased fluorescence arising from enhanced green fluorescent protein (EGFP)-cPLA2␣ in the forming phagosome was due to an increased volume of cytoplasm imaged in the plane
Because it is well described that PIP2 increases in the phagosome [31], we studied the behavior of the 4-Lys mutant (EGFP-4KE/A-cPLA2␣) in macrophages exposed to opsonized zymosan

Summary

Introduction

Group V-secreted phospholipase A2 and group VIA calcium-independent phospholipase A2 were studied, but the results indicated that neither of these translocated to the phagosome These data indicate that the polybasic cluster of cPLA2␣ (Lys488/Lys541/Lys543/Lys544) regulates the subcellular localization of the enzyme in intact cells under physiologically relevant conditions.–—Casas, J., M. Free AA will in turn be oxygenated by specific enzymes to generate a wide variety of compounds with potent pro- and anti-inflammatory actions, collectively called the eicosanoids [6, 7]. Some of these compounds have been described as important bactericidal agents [8]. The catalytic domain contains a cluster of cationic amino acids (Lys488, Lys541, Lys543, and Lys544) that may mediate

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Conclusion