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

doi:10.1194/jlr.M001461

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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 Balsinde¹^,*^, and María A. Balboa¹^,*^,

^* Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas (CSIC),47003 Valladolid, Spain
Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain

¹ To whom correspondence should be addressed. e-mail: jbalsinde{at}ibgm.uva.es; mbalboa{at}ibgm.uva.es

Group IVA cytosolic phospholipase A₂ ${alpha}$ (cPLA₂ ${alpha}$ ) plays a role inthe microbicidal machinery of immune cells by translocatingto phagosomes to initiate the production of antimicrobial eicosanoids.In this work, we have studied the involvement of the cationiccluster of cPLA₂ ${alpha}$ (Lys⁴⁸⁸/Lys⁵⁴¹/Lys⁵⁴³/Lys⁵⁴⁴) in the translocationof the enzyme to the phagosomal cup in human macrophages respondingto opsonized zymosan. Phagocytosis was accompanied by an increasedmobilization of free arachidonic acid, which was strongly inhibitedby pyrrophenone. In transfected cells, a catalytically activeenhanced green fluorescent protein-cPLA₂ ${alpha}$ translocated to thephagocytic cup, which was corroborated by frustrated phagocytosisexperiments using immunoglobulin G-coated plates. However, acPLA₂ ${alpha}$ mutant in the polybasic cluster that cannot bind the anionicphospholipid phosphatidylinositol 4, 5-bisphosphate (PIP₂) didnot translocate to the phagocytic cup. Moreover, an enhancedyellow fluorescent protein (EYFP)-cPLA₂ ${alpha}$ and an enhanced cyanfluorescent protein-pleckstrin homology (PH) domain of the phospholipaseC ${delta}$ 1 (PLC ${delta}$ ₁) construct that specifically recognizes endogenousPIP₂ in the cells both localized at the same sites on the phagosome.High cellular expression of the PH domain inhibited EYFP-cPLA₂ ${alpha}$ translocation. On the other hand, group V-secreted phospholipaseA₂ and group VIA calcium-independent phospholipase A₂ were alsostudied, but the results indicated that neither of these translocatedto the phagosome. Collectively, these data indicate that thepolybasic cluster of cPLA₂ ${alpha}$ (Lys⁴⁸⁸/Lys⁵⁴¹/Lys⁵⁴³/Lys⁵⁴⁴) regulatesthe subcellular localization of the enzyme in intact cells underphysiologically relevant conditions.

Supplementary key words lipid mediators • monocytes/macrophages • phagocytosis • phospholipase A₂ • inflammation

Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone; bis-BODIPY FL C11-PC, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-sindecane-3-undecanoyl)-sn-glycero-3-phosphocholine; cPLA₂ ${alpha}$ , group IVA cytosolic phospholipase A₂; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; iPLA₂, calcium-independent phospholipase A₂; PIP₂, phosphatidylinositol 4,5-bisphosphate; PLA₂, phospholipase A₂; PLC ${delta}$ ₁, phospholipase Cdelta1; PLC ${delta}$ ₁-PH, pleckstrin homology domain of PLC ${delta}$ ₁; sPLA₂, secreted phospholipase A₂; sPLA₂-V, group V secreted phospholipase A₂

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