Phospholipase A2 hydrolyzes the sn-2 ester bond of glycerophospholipids that produce free fatty acids and lysophospholipids. Cytosolic phospholipase A2s (cPLA2, group IV) are a subgroup of enzymes that act on the intracellular phospholipid membrane. The best investigated cPLA2α (group IVA) is a key enzyme for lipid mediator production in vivo. Here we report cloning and characterization of novel murine cPLA2s: cPLA2δ (group IVD), cPLA2ϵ (group IVE), and cPLA2ζ (group IVF), that form a gene cluster with cPLA2β (group IVB). The deduced amino acid sequences of cPLA2δ, ϵ, and ζ demonstrated a conserved domain structure of cPLA2, i.e. one C2 domain and one lipase domain. The potential catalytic dyad, Ser and Asp, was conserved for these newly cloned cPLA2s along with relatively high conservation for the surrounding residues. Transcripts of murine cPLA2δ, ϵ, and ζ appeared to be enriched in certain organs rather than ubiquitous distribution. Major Northern signals for cPLA2δ were detected in placenta, cPLA2ϵ in thyroid, heart, and skeletal muscle, and cPLA2ζ in thyroid. Recombinant proteins expressed in human embryonic kidney 293 cells demonstrated molecular sizes of about 100 kDa by Western blotting and exhibited Ca2+-dependent PLA2 activities on 1-palmitoyl-2-[14C]arachidonoyl-phosphatidylcholine substrate. In contrast to cPLA2α, cPLA2ζ preferred phosphatidylethanolamine to phosphatidylcholine. Intracellular localization was visualized by green fluorescent-tagged proteins. Each molecule showed specific localization, and cPLA2δ translocated from the cytosol to the perinuclear region by calcium-ionophore stimulation. We thus discovered these functional novel cPLA2 genes, which cluster on murine chromosome 2E5.
This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. S. and N. U.), and a grant from the ONO Medical Research Foundation (to N. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) AB195276 for cPLA2δ, AB195277 for cPLA2ϵ, and AB195278 for cPLA2ζ.
