Identification of hepatic peroxisomal phospholipase A₂and characterization of arachidonic acid‐containing choline glycerophospholipids in hepatic peroxisomes

Abstract

Recently, a sequence encoding a novel mammalian calcium-independent phospholipase A 2 (iPLA 2 Q Q) was identi¢ed in the human genome and subsequently cloned and expressed in Sf9 insect cells. Unexpectedly, expression studies in recombinant systems demonstrated the usage of multiple translation initiation codons resulting in di¡erent polypeptides. Herein, we demonstrate that hepatic iPLA 2 Q Q is localized to rat liver peroxisomes, possesses a molecular mass of 63 kDa and that peroxisomal membranes are highly enriched in arachidonic acid-containing phospholipids. Collectively, these results provide the ¢rst demonstration of iPLA 2 Q Q in mammalian tissue and suggest the possibility that iPLA 2 Q Q can contribute to lipid second messenger generation by hydrolysis of peroxisomal arachidonic acid-containing phospholipids. ß

Group VIB Ca2+-independent Phospholipase A2γ Promotes Cellular Membrane Hydrolysis and Prostaglandin Production in a Manner Distinct from Other Intracellular Phospholipases A2

“…As reported previously in insect Sf9 cells (33), iPLA 2 ␥ has four potential translation initiation sites, which produce distinct sizes of its protein. Our immunoblot analysis revealed that the 63-kDa form, the translation of which is likely to be initiated at the fourth methionine, was expressed mainly in HEK293, HCA-7, and WiDr cells, whereas the full-size 88-kDa form was dominant in BEAS-2B and 3Y1 cells (Fig.…”

“…This suggests that the regulatory functions (and their underlying mechanisms) of iPLA 2 ␥ are distinct from those of iPLA 2 ␤. Although some unique features of iPLA 2 ␥ have been recently reported, such as stable association with membranes and utilization of distinct translation initiation sites producing different sizes of enzyme (31)(32)(33), its cellular functions remain largely unknown. More recently, novel members of the iPLA 2 family, including iPLA 2 ␦, which displays lysophospholipase ac-tivity rather than PLA 2 activity (34), and iPLA 2 ⑀, iPLA 2 , and iPLA 2 , which exhibit triacylglycerol lipase and transacylase activities (35), have been identified.…”

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets

“…Human iPLA 2 -γ has multiple alternative start sites for translation resulting in functional PLA 2 γ proteins with MW of 63-, 74-, 77-and 88-kDa [7]. Initially, the subcellular localization of iPLA 2 γ was assumed to be peroxisomal due to the presence of a C-terminal-SKL sequence [7] and recent studies have confirmed that the peroxisomal iPLA 2 γ is the 63-kDa protein [20,21]. In contrast, other recent studies have demonstrated higher molecular weight forms of iPLA 2 γ are present in rat heart mitochondria [22] and we have demonstrated that the 88-kDa iPLA 2 γ isoform is present in rabbit heart and kidney ER and mitochondria [23].…”

Complement-mediated Activation of Calcium-independent Phospholipase A2γ

“…Indeed, a previous study showed that iPLA 2 ␥ contains four N-terminal methionine residues that may act as translation initiation sites, resulting in 88-, 77-, 74-, and 63-kDa forms of iPLA 2 ␥ in SF9 insect cells (33). To determine if the N-terminal region may be involved in localization and/or regulation of iPLA 2 ␥ catalytic activity, we deleted the 220 N-terminal amino acids (spanning between the 1st and 4th methionine) to generate a short form of iPLA 2 ␥ in which the GFP-iPLA 2 ␥ fusion would be at the 4th methionine (M4 GFPiPLA 2 ␥).…”

“…These distinct sites of localization may be a result of specific domains in the structure of the enzyme (32). iPLA 2 ␥ gene transcription and translation appear complex, as distinct translation initiation sites, resulting in the production of 88-, 77-, 74-, and 63-kDa forms of the enzyme were reported (33). iPLA 2 ␥ contains a consensus site for nucleotide binding and a lipase consensus motif in its C-terminal half as well as potential cAMPdependent protein kinase, protein kinase C, and extracellular signal-regulated kinase (ERK) phosphorylation sites (32).…”