Multiple Splice Variants of the Human Calcium-independent Phospholipase A2 and Their Effect on Enzyme Activity

Abstract

Recently, the cloning of a novel Ca 2؉ -independent phospholipase A 2 (iPLA 2 ) from Chinese hamster ovary cells as well as from mouse and rat sources containing a C-terminal lipase motif and eight N-terminal ankyrin repeats has been described. In this report we describe the cloning of the human iPLA 2 cDNA and its expression in B-cells and show that the iPLA 2 gene undergoes extensive alternative splicing generating multiple isoforms that contribute to a novel mechanism to control iPLA 2 activity. The full-length cDNA clone encodes a 806-amino acid protein with a calculated molecular mass of 88 kDa. The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hamster and rodent forms of the enzyme but has an additional 54-amino acid proline-rich insertion in the last of the eight ankyrin repeats (residues 395-449). Furthermore, at least three additional isoforms most likely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar to iPLA 2 but terminates just after the lipase active site, and two other isoforms contain only the iPLA 2 ankyrin repeat sequence (ankyrin-iPLA 2 -1 and -2). Ankyrin repeats are involved in protein-protein interactions and because the purified iPLA 2 enzyme exists as a multimeric complex of 270 -350 kDa, the expression of just the ankyrin-iPLA 2 sequence suggested that these may also interact with the iPLA 2 oligomeric complexes and perhaps modulate PLA 2 activity. Transfection of the human iPLA 2 cDNA into COS cells resulted in a substantial increase in calcium-independent PLA 2 activity in cell lysate. No activity above background was observed following ankyrin-iPLA 2 -1 cDNA transfection. However, co-transfection of the ankyrin-iPLA 2 -1 and the iPLA 2 cDNAs resulted in a 2-fold reduction in activity compared with iPLA 2 alone. A similar co-transfection of ankyrin-iPLA 2 -1 cDNA with the cPLA 2 cDNA had no effect on PLA 2 activity. These results suggest that the ankyrin-iPLA 2 sequence can function as a negative regulator of iPLA 2 activity and that the alternative splicing of the iPLA 2 gene can have a direct effect on the attenuation of enzyme activity.

Cell Cycle Dependence of Group VIA Calcium-independent Phospholipase A2 Activity

“…800 ng of total RNA was reverse-transcribed using Thermoscript reverse transcriptase (Invitrogen) and subjected to 30 cycles of PCR amplification (95°C for 1 min, 55°C for 45 s, and 72°C for 1 min). The iPLA 2 -specific primers used were 5Ј-cagggctctgcagcgccacatcat-3Ј (forward) and 5Ј-ggccctctcgatggcgatgaggag-3Ј (reverse) and they generated 237, 290, and 342 base pair products as expected based on Larsson et al (12). Quantitative real-time PCR was employed to determine the levels of iPLA 2 and ankyrin-iPLA 2 transcripts.…”

“…A recent report suggests that the catalytic activity of the enzyme is modulated by catalytically inactive truncated iPLA 2 proteins that are encoded by alternatively spliced mRNAs (12). These splice variants are referred to as ankyrin-iPLA 2 s because the alternative splicing is predicted to generate truncated proteins that retain the N-terminal ankyrin repeats but lack the C-terminal lipase site.…”

FcγRI-triggered Generation of Arachidonic Acid and Eicosanoids Requires iPLA2 but Not cPLA2 in Human Monocytic Cells

“…Whereas Group VI PLA 2 , or iPLA 2 , are Ca 2ϩ -independent enzymes (10,11), also possessing a catalytic serine, yet its structure is far distant from that of the cPLA 2 family. iPLA 2 occurs in multiple alternative splicing variants, the majority of which are enzymatically functional (12,13). Mammalian iPLA 2 s are classified as groups VIA and VIB, (iPLA 2 ␤ and iPLA 2 ␥, respectively) (10).…”

A Requirement for Calcium-independent Phospholipase A2 in Thrombin-induced Arachidonic Acid Release and Growth in Vascular Smooth Muscle Cells

“…Phospholipase A 2 s (PLA 2 s) are the major rate-limiting enzymes in the release of arachidonic acid in many cell types (11,20,21). Among the growing number of PLA 2 s that have been isolated and characterized thus far, a calcium-dependent high molecular mass cytosolic PLA 2 (cPLA 2 ) and a calcium-independent PLA 2 (iPLA 2 ) have been shown to play an important role in arachidonic acid release in response to a number of stimulants including receptor tyrosine kinase and G protein-coupled receptor agonists (20 -25).…”