MULTIPLE SPLICE VARIANTS OF THE HUMAN CALCIUM-INDEPENDENT PHOSPHOLIPASE A(2) AND THEIR EFFECT ON ENZYME-ACTIVITY

Recently, the cloning of a novel Ca2+-independent phospholipase A(2) ( iPLA(2)) from Chinese hamster ovary cells as well as from mouse and ra t sources containing a C-terminal lipase motif and eight N-terminal an kyrin repeats has been described. In this report we describe the cloni ng of the human iPLA(2) cDNA and its expression in B-cells and show th at the iPLA(2) gene undergoes extensive alternative splicing generatin g multiple isoforms that contribute to a novel mechanism to control iP LA(2) activity. The full-length cDNA clone encodes a 806-amino acid pr otein with a calculated molecular mass of 88 kDa, The protein contains a lipase motif, GXSXG, and ankyrin repeats, as described for the hams ter and rodent forms of the enzyme but has an additional 54-amino acid proline rich insertion in the last of the eight ankyrin repeats (resi dues 395-449). Furthermore, at least three additional isoforms most li kely due to alternative splicing were identified. One that is present as a partial cDNA in the expressed sequence tag data base is similar t o iPLA(2) but terminates just after the lipase active site, and two ot her-isoforms contain only the iPLA(2) ankyrin repeat sequence (ankyrin -iPLA(2)-1 and -2), Ankyrin repeats are involved in protein-protein in teractions and because the purified iPLA(2) enzyme exists as a multime ric complex of 270-350 kDa, the expression of just the ankyrin-iPLA(2) sequence suggested that these may also interact with the iPLA(2) olig omeric complexes and perhaps modulate PLA(2) activity. Transfection of the human iPLA(2) cDNA into COS cells resulted in a substantial incre ase in calcium-independent PLA(2) activity in cell lysate, No activity above background was observed following ankyrin-iPLA(2)-1 cDNA transf ection. However, co-transfection of the ankyrin-iPLA(2)-1 and the iPLA (2) cDNAs resulted in a 2-fold reduction in activity compared with iPL A(2) alone, A similar co-transfection of ankyrin-iPLA(2)-1 cDNA with t he cPLA(2) cDNA had no effect on PLA(2) activity, These results sugges t that the ankyrin-iPLA(2) sequence can function as a negative regulat or 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.