Abstract
The subcellular location of the enzymes of eicosanoid biosynthesis is critical for their co-ordinate action in the generation of leukotrienes and prostaglandins. This activity is thought to occur predominantly at a perinuclear location. Whereas the subcellular locations of cytosolic phospholipase (PL) A(2) and each of the pathway enzymes of eicosanoid generation have been defined, the distribution of the low molecular weight species of PLA(2) has remained elusive because of the lack of antibodies that distinguish among homologous family members. We have prepared affinity-purified rabbit antipeptide IgG antibodies that distinguish mouse group IIA PLA(2) and group V PLA(2). Immunofluorescence staining and immunogold electron microscopy reveal different subcellular locations for the enzymes. Group IIA(2) PLA(2) is present in the secretory granules of mouse bone marrow-derived mast cells, consistent with its putative role in facilitating secretory granule exocytosis and its consequent extracellular action. In contrast, group V PLA(2) is associated with various membranous organelles including the Golgi apparatus, nuclear envelope, and plasma membrane. The perinuclear location of group V PLA(2) is consistent with a putative interaction with translocated cytosolic PLA(2) in supplying arachidonic acid for generation of eicosanoid products, while the location in Golgi cisternae may also reflect its action as a secreted enzyme. The spatial segregation of group IIA PLA(2) and group V PLA(2) implies that these enzymes are not functionally redundant.
Highlights
The subcellular location of the enzymes of eicosanoid biosynthesis is critical for their co-ordinate action in the generation of leukotrienes and prostaglandins
Evidence is accumulating that the cellular functions of the enzymes involved in leukotriene and prostaglandin biosynthesis are regulated in part by their induced expression (PGHS-2), subcellular localization (FLAP, LTC4 synthase, prostaglandin endoperoxide synthase (PGHS)-1, PGHS-2), and translocation (5-LO and cytosolic PLA2 (cPLA2))
Transcripts for group IIA PLA2 are present in bone marrow-derived mast cells (BMMC) from BALB/c mice [18, 32], BMMC from mice genetically deficient in group IIA PLA2 are not impaired in their ability to generate eicosanoids even though secretory granule exocytosis is somewhat attenuated [17, 18]
Summary
Chemicals were purchased from Sigma unless otherwise noted. Restriction endonucleases were purchased from Roche Molecular Biochemicals. The primers, PIB-1 (5Ј-GGCTGTGTGGCAGTTCCGC-3Ј) and PIB-2 (5Ј-GTGTTGGTGTAGGGGTTGTC-3Ј), corresponding to nucleotides 77–95 and 273– 292, respectively, of the rat sequence, were used in a 25-cycle PCR reaction with the mouse lung cDNA as a template at an annealing temperature of 55 °C. Expression of Group IIA PLA2—The cDNA encoding the open reading frame of mouse group IIA PLA2 [18] was subcloned into pVL1393 (PharMingen, San Diego, CA) and co-transfected with Baculo-Gold linearized baculovirus DNA (PharMingen) into Sf9 cells with calcium phosphate according to the manufacturer’s instructions. Fractions containing PLA2 activity were resolved in 12% SDS-PAGE (Novex) under reducing conditions and silver-stained according to manufacturer’s instructions (Sigma) to reveal a single protein band of ϳ17 kDa. Total RNA was extracted from mouse BMMC, heart, and lung, and cDNA was synthesized with MMLV reverse transcriptase as described above. The protein bands were visualized with enhanced chemiluminescence (SuperSignal®, Pierce) and Biomax XR film (Eastman Kodak Co.) for 30 s to 5 min
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