Enzymatic Properties of Human Cytosolic Phospholipase A2γ

Allison Stewart,Moumita Ghosh,Diane M Spencer,Christina C Leslie

doi:10.1074/jbc.m204856200

Abstract

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.

Highlights

Mammalian cells contain structurally diverse phospholipase A2 (PLA2)1 enzymes that differ in their location and regulation [1,2,3]
There was no immunoreactive material detected with this antibody in Sf9 cells expressing cytosolic PLA2s (cPLA2)␣ or in cells infected with vector control virus
As previously reported for cPLA2␥ expressed in Chinese hamster ovary cells [40], cPLA2␥ expressed in Sf9 cells was primarily found in the membrane fraction when evaluated both by assaying

Summary

The abbreviations used are

PLA2␥, phospholipase A2␥; cPLA2␥, cytosolic phospholipase A2␥; cPLA2␣, cytosolic phospholipase A2␣; cPLA2␤, cytosolic phospholipase A2␤; Sf9, S. frugiperda; MAFP, methylarachidonyl fluorophosphonate; BEL, (E)-6-(bromoethylene)-3-(1naphthalenyl)-2H-tetrahydropyran-2-one; GFP, green fluorescent protein; His6, 6-histidine tag; m.o.i., multiplicity of infection; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; MEM, minimum essential medium; FBS, fetal bovine serum; HEK, human embryonic kidney; PLD2, phospholipase D2; PMA, phorbol 12-myristate 13-acetate; iPLA2, calcium-independent phospholipase A2; sPLA2, secreted phospholipase A2; Ni-NTA, nickel-nitrilotriacetic acid. Numerous intracellular PLA2 enzymes have been identified and can be subdivided into two major families, the group IV cytosolic PLA2s (cPLA2) and the group VI calcium-independent (iPLA2) forms. The calcium-independent cPLA2␥ lacks a C2 domain and is membrane-bound It contains a C-terminal prenylation site and potential acylation sites. CPLA2␥ is expressed at high levels in heart and skeletal muscle [40] It lacks the mitogen-activated protein kinase consensus site but contains potential protein kinase C phosphorylation sites. When overexpressed in lung fibroblasts, cPLA2␥ is activated by serum resulting in release of unsaturated and saturated fatty acids, unlike cPLA2␣, which primarily releases arachidonic acid

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION