The human calcium-independent phospholipase A2 gene -: Multiple enzymes with distinct properties from a single gene

Recently, we reported the human 88-kDa calcium-independent phospholipase A(2) (iPLA(2)) cDNA sequence, as well as extensive alternative splicing of the iPLA(2) mRNA. In this report we identified the gene coding for iPLA(2), which was localized on chromosome 22q13.1. The gene consists of at least 17 exons spanning > 69 kb. Based on the iPLA2 gene organization the splice variants can be explained. The putative promotor for the iPLA(2) gene lacks a TATA-box and contains a CpG island as well as several potential Sp-1-binding sites. Furthermore, the 5'-flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence. Northern blot analysis of iPLA(2) mRNA in various human tissues demonstrated tissue-specific expression of four distinct iPLA(2) transcripts. The native human 3.2-kb iPLA(2) transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow. Studies on the subcellular localization of the native iPLA(2) protein were performed in COS-7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA(2) contained equal amounts of calcium-independent PLA(2) activity. However, the membrane fraction displayed a 5.5-fold increased activity in iPLA2 overexpressing cells. This increased calcium-independent PLA(2) activity correlated with the presence of iPLA(2) immunoreactive protein in the membrane fraction, indicating that this form of iPLA(2) protein was membrane associated. Studies of iPLA(2) in rat vascular smooth muscle cells verified the membrane association of this form of iPLA(2). The major difference between this form of iPLA(2) enzyme and the soluble forms of iPLA(2) studied previously is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane-associated protein. In summary, these results demonstrate that alternative splicing of the human iPLA(2) transcript generates multiple iPLA(2) isoforms with distinct tissue distribution and cellular localization.