Identification of the Expressed Form of Human Cytosolic Phospholipase A2β (cPLA2β)

Abstract

In this study, we identify the principal splice variant of human cytosolic phospholipase A 2 ␤ (cPLA 2 ␤) (also known as Group IVB cPLA 2 ) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA 2 ␤ is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA 2 ␤. BEAS-2B cells contain three different RNA splice variants of cPLA 2 ␤ (␤1, ␤2, and ␤3). cPLA 2 ␤1 is identical to the previously cloned cPLA 2 ␤, predicted to encode a 114-kDa protein. However, cPLA 2 ␤2 and cPLA 2 ␤3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA 2 ␤ in BEAS-2B cells is the translated product of cPLA 2 ␤3. cPLA 2 ␤3 exhibits calcium-dependent PLA 2 activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA 2 ␣, cPLA 2 ␤3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA 2 ␤3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.Phospholipase A 2 (PLA 2 ) 2 enzymes catalyze hydrolysis of sn-2 acyl chains from membrane phospholipids. They execute diverse functions, such as digestion of dietary phospholipids, microbial degradation, membrane remodeling, and production of lipid mediators. Traditionally, PLA 2 s are grouped depending on their active site residues, requirements for calcium, and localization in the cell. Three main classes of PLA 2 s are Group VI intracellular calcium-independent PLA 2 s, low molecular weight secreted PLA 2 s, and Group IV cytosolic PLA 2 s (1, 2). Group IVA cytosolic PLA 2 ␣ has received special attention, because it is the only PLA 2 that selectively hydrolyzes arachidonic acid from the sn-2 position of membrane phospholipids (1). Arachidonic acid is the precursor of prostaglandins and leukotrienes (1, 3). Mice genetically deficient in cPLA 2 ␣ have provided evidence for its critical role in regulating physiological processes and various diseases (4 -11). cPLA 2 ␣ contains an N-terminal calcium binding domain (C2 domain) and a C-terminal catalytic domain (12). Calcium binds to the C2 domain and facilitates the translocation of the enzyme from cytosol to the Golgi, endoplasmic reticulum, and nuclear envelope (13-16). Five other members of the Group IV cPLA 2 family, cPLA 2 ␤ (Group IVB), cPLA 2 ␥ (Group IVC), cPLA 2 ␦ (Group IVD), cPLA 2 (Group IVE), and cPLA 2 (Group IVF), have been identified (17-21). cPLA 2 ␦, -, and -are clustered near cPLA 2 ␤ on mouse chromosome 2 and have more homology to cPLA 2 ␤ than to cPLA 2 ␣ or cPLA 2 ␥ (21). From analysis of the human genome, cPLA 2 ␤ is similarly positioned near cPLA 2 ␦, -, and -on chromosome 15. All members of the Group IV family have a conserved Ser/Asp dyad necessary for catalysis (12).Human cPLA 2 ␤ was originally cloned from human brain ...

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

“…The similar inhibitor sensitivity of cPLA 2 and cPLA 2 ␣ is in contrast to the results with mouse cPLA 2 ␤, which is not inhibited by pyrrolidine-2 and Wyeth-1. Human cPLA 2 ␤ and human cPLA 2 ␥ also are not inhibited by pyrrolidine-2 and pyrrolidine-1, respectively (22,39). The basis for the differences in sensitivity of the GIV PLA 2 family to pyrrolidine-2 and Wyeth-1 is not clear at this time.…”

“…We recently reported that human cPLA 2 ␤ also has low enzymatic activity and that neither mouse nor human cPLA 2 ␤ is inhibited by cPLA 2 ␣ inhibitors (22). Analysis of the mouse cPLA 2 ␤ transcript present in lung fibroblasts demonstrates that it does not contain the truncated JmjC domain found in the N terminus of human cPLA 2 ␤.…”

“…The C2 and catalytic domains of mouse cPLA 2 ␤ have 82% amino acid identity with human cPLA 2 ␤1. We have recently identified three transcripts of human cPLA 2 ␤ in BEAS-2B lung epithelial cells (22). cPLA 2 ␤1 is identical to the form originally cloned (14,21), and cPLA 2 ␤2 and cPLA 2 ␤3 contain internal deletions in the catalytic domain (22).…”

“…It contains a novel, N-terminal-truncated JmjC domain immediately upstream of the C2 domain. We have recently found that the principle form of cPLA 2 ␤ translated in human cells is a novel splice variant (cPLA 2 ␤3) that contains an internal deletion in the catalytic domain (22). 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 ␣.…”

“…Human cPLA 2 ␦ is associated with psoriatic lesions and is expressed in stratified squamous epithelium (19). Human cPLA 2 ␤ is widely expressed and occurs as multiple splice variants (14,21,22). It contains a novel, N-terminal-truncated JmjC domain immediately upstream of the C2 domain.…”