title: The human calcium-independent phospholipase A2 gene multiple enzymes with distinct properties from a single gene.
abstract: Recently, we reported the human 88-kDa calcium-independent phospholipase A2 (iPLA2) cDNA sequence, as well as extensive alternative splicing of the iPLA2 mRNA. In this report we identified the gene coding for iPLA2, 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 iPLA2 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 iPLA2 mRNA in various human tissues demonstrated tissue-specific expression of four distinct iPLA2 transcripts. The native human 3.2-kb iPLA2 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 iPLA2 protein were performed in COS-7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA2 contained equal amounts of calcium-independent PLA2 activity. However, the membrane fraction displayed a 5.5-fold increased activity in iPLA2 overexpressing cells. This increased calcium-independent PLA2 activity correlated with the presence of iPLA2 immunoreactive protein in the membrane fraction, indicating that this form of iPLA2 protein was membrane associated. Studies of iPLA2 in rat vascular smooth muscle cells verified the membrane association of this form of iPLA2. The major difference between this form of iPLA2 enzyme and the soluble forms of iPLA2 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 iPLA2 transcript generates multiple iPLA2 isoforms with distinct tissue distribution and cellular localization.
authors.affiliation: Department of Medical Biochemistry, Karolinska Institutet, Stockholm, Sweden.
authors.forename: P K
authors.initials: PK
authors.lastname: Larsson Forsell
authors.forename: B P
authors.initials: BP
authors.lastname: Kennedy
authors.forename: H E
authors.initials: HE
authors.lastname: Claesson
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D004247:DNA; D005091:Exons; D006801:Humans; D007438:Introns; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2