Identification of Novel Cytosolic Phospholipase A2s, Murine cPLA2δ, ϵ, and ζ, Which Form a Gene Cluster with cPLA2β

Abstract

Phospholipase A 2 hydrolyzes the sn-2 ester bond of glycerophospholipids that produce free fatty acids and lysophospholipids. Cytosolic phospholipase A 2 s (cPLA 2 , group IV) are a subgroup of enzymes that act on the intracellular phospholipid membrane. The best investigated cPLA 2 ␣ (group IVA) is a key enzyme for lipid mediator production in vivo. Here we report cloning and characterization of novel murine cPLA 2 s: cPLA 2 ␦ (group IVD), cPLA 2 ⑀ (group IVE), and cPLA 2 (group IVF), that form a gene cluster with cPLA 2 ␤ (group IVB). The deduced amino acid sequences of cPLA 2 ␦, ⑀, and demonstrated a conserved domain structure of cPLA 2 , i.e. one C2 domain and one lipase domain. The potential catalytic dyad, Ser and Asp, was conserved for these newly cloned cPLA 2 s along with relatively high conservation for the surrounding residues. Transcripts of murine cPLA 2 ␦, ⑀, and appeared to be enriched in certain organs rather than ubiquitous distribution. Major Northern signals for cPLA 2 ␦ were detected in placenta, cPLA 2 ⑀ in thyroid, heart, and skeletal muscle, and cPLA 2 in thyroid. Recombinant proteins expressed in human embryonic kidney 293 cells demonstrated molecular sizes of about 100 kDa by Western blotting and exhibited Ca 2؉ -dependent PLA 2 activities on 1-palmitoyl-2-[ 14 C]arachidonoyl-phosphatidylcholine substrate. In contrast to cPLA 2 ␣, cPLA 2 preferred phosphatidylethanolamine to phosphatidylcholine. Intracellular localization was visualized by green fluorescent-tagged proteins. Each molecule showed specific localization, and cPLA 2 ␦ translocated from the cytosol to the perinuclear region by calcium-ionophore stimulation. We thus discovered these functional novel cPLA 2 genes, which cluster on murine chromosome 2E5.

Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2ζ in Mouse Lung Fibroblasts

“…Our data indicates that this is due to the calcium-dependent activation of cPLA 2 . The cPLA 2 transcript is highly expressed in mouse thyroid and at lower levels in stomach, large intestine, and prostate (20). The mRNA for cPLA 2 was not detected in whole mouse lung; however, the lung comprises over 20 different cell types, and cell type-specific expression of cPLA 2 is possible.…”

“…In contrast separate adjacent genes on mouse chromosome 2 encode a complete JmjC domain and cPLA 2 ␤. Both mouse and human cPLA 2 ␤ are similarly situated near the gene cluster encoding cPLA 2 ⑀, -␦, and - (4,20). cPLA 2 ␤ has very low enzymatic activity when assayed in vitro; however, it is possible that it has a unique substrate and/or cofactors in vivo.…”

“…In contrast, the mouse cPLA 2 ␥ homologue is only 50% homologous to human cPLA 2 ␥ and is expressed exclusively in oocytes (18). cPLA 2 ␦, cPLA 2 ⑀, and cPLA 2 form a gene cluster near cPLA 2 ␤ in humans and mice and have more homology to cPLA 2 ␤ than to cPLA 2 ␣ (4,19,20). Human cPLA 2 ␦ is associated with psoriatic lesions and is expressed in stratified squamous epithelium (19).…”

“…cPLA 2 ␤3 exhibits calcium-dependent PLA 2 activity but is constitutively bound to mitochondria and early endosomes in cells, suggesting a mechanism of regulation and function distinct from cPLA 2 ␣. cPLA 2 ␦, cPLA 2 ⑀, and cPLA 2 have been cloned from mouse tissues; however, only preliminary information is available about their biochemical properties, and nothing is known of their functional roles (20).…”

Phospholipase A2IVα Regulates Phagocytosis Independent of Its Enzymatic Activity

“…The PLA 2 family includes several relatively unrelated proteins, among which are the following: the low molecular weight secreted enzymes (sPLA 2 s), which have been implicated in eicosanoid generation, inflammation, host defense, and atherosclerosis (5); the cytosolic Ca 2ϩ -independent group VI PLA 2 s, which have been reported to have roles in phospholipid homeostasis and membrane remodeling (6); and the group IV cytosolic PLA 2 s, which include six high molecular mass enzymes in Mus musculus (as PLA 2 IV␣ to PLA 2 IV), with three newly cloned isoforms (7). PLA 2 IV␣ has an essential role in initiation of the arachidonate pathway, and it is mainly involved in inflammation, intestinal ulceration, acute lung injury, anaphylaxis, and parturition, as shown through the PLA 2 IV␣ knockout mouse approach (8 -10).…”