The measurement of lysosomal phospholipase A2 activity in plasma

Abstract

This article is available online at http://www.jlr.org named lysosomal phospholipase A2 (LPLA2) ( 2, 3 ). The enzyme purifi ed from calf brain is a water-soluble glycoprotein consisting of a single polypeptide chain with a molecular weight of 45 kDa with Ca 2+ -independent phospholipase A2 activity at a pH optimum of 4.5 ( 2 ). LPLA2 belongs to the ␣ ␤ -hydrolase super-family of enzymes and has 49% protein sequence identity to lecithin cholesterol acyltransferase, with the latter thought to have arisen as a gene duplication product ( 3 ). LPLA2 is now classifi ed as the only member of the group XV phospholipase A2 family ( 4 ).LPLA2 in mouse and rat is highly expressed in alveolar macrophages and may be centrally involved in the catabolism of glycerophospholipids in pulmonary surfactant ( 5 ). Additionally, both alveolar and peritoneal macrophages obtained from LPLA2 knockout mice are characterized by the significant accumulation of phospholipids and the formation of numerous cytoplasmic multi-lamellar inclusion bodies, a hallmark of cellular phospholipidosis ( 6 ). The phospholipid accumulation found in alveolar macrophages obtained from LPLA2 knockout mice is markedly reduced by the addition of recombinant mouse LPLA2 ( 7 ). The recombinant LPLA2 is taken into the cells via mannose receptors and translocated into acidic compartments such as late endosomes and lysosomes ( 7 ). In addition, endogenous LPLA2 in the alveolar macrophage is secreted following stimulation with zymosan, indicating that LPLA2 is a secreted enzyme as well as a lysosomal enzyme. Recently, we have demonstrated that negatively charged lipids mediate LPLA2 membrane binding through an electrostatic interaction and amplify apparent LPLA2 activity ( 8 ). These observations indicate that cationic amphiphilic drug-induced phospholipidosis in some cases Abstract A defi ciency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially refl ect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/ N -acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specifi city of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfl uorophosphonate. The inhibitor also suppressed the...

The phospholipase A2 activity of peroxiredoxin 6 [S]

“…LPLA 2 also is expressed in alveolar macrophages, and this, rather than aiPLA 2 , may be the major macrophage-related phospholipid degradative enzyme (97,98).…”

Preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase A2

“…9E, F). LPLA2 is a secreted protein as well as a lysosomal protein (28)(29)(30). LPLA2 activity in the CHO/LPLA2 cell-cultured medium after a 4 h treatment with or without PAzPC was three times higher than that after a 1 h treatment (data not shown).…”

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

“…In addition, older LPLA2 Ϫ / Ϫ mice present similar symptoms to human autoimmune disease ( 7 ). Like other lysosomal enzymes, LPLA2 is secreted to extracellular space ( 8,9 ). Recently, LPLA2 activity was found in the aqueous humor as well as ocular tissues in pig eye ( 10 ).…”

“…Therefore, such a transacylation reaction has been used to detect the LPLA2 activity in the cell, tissue, and extracellular fl uid ( 3,6,9,10 ). In the present study, to measure the transacylase and phospholipase A2 activity of LPLA2 in the transfectants, DOPC/ sulfatide/NAS liposomes were incubated with cell extracts obtained from individual transfectants under acidic conditions.…”