Patatin-like Phospholipase Domain Research Articles

A phospholipase A2‐linked receptor for PEDF

Pigment epithelium-derived factor (PEDF), a 50-kDa extracellular serpin, is a neurotrophic and antiangiogenic factor for the retina. Its biological functions are independent from its serine protease inhibition potential. Previously we provided evidence for high affinity PEDF-binding sites and proteins in retina cell-surfaces. Recently a novel human retinal pigment epithelial gene was identified as potential PEDF receptor and termed PEDF-R (pseudonyms: PNLP2, TTS-2.1, ATGL, iPLAζ, desnutrin). It codes for a polypeptide (504 amino acids) with a patatin-like phospholipase (PLA2) domain, and 4 transmembrane, 3 intracellular and 2 extracellular regions. RT-PCR and immunohistochemistry confirmed the PEDF-R expression in the retina. The potential PLA2 activity of PEDF-R was investigated. Recombinant epitope-tagged PEDF-R polypeptide was synthesized in cell-free systems and purified. Using dilinoleoyl phosphatidylcholine as substrate, PEDF-R exhibited potent PLA activity, which was optimum at pH 7.5. Surface plasmon resonance revealed high affinity for the PEDF:PEDF-R interactions (KD = ~3 nM), which decreased significantly with other serpins (maspin, ovalbumin). Preincubation of PEDF-R with increasing concentrations of PEDF stimulated the PLA activity of PEDF-R, but the stimulation did not occur when BSA replaced PEDF. PEDF alone did not show activity, and did not stimulate hog pancreas PLA2. These results demonstrate that the recombinant PEDF-R exhibited PLA activity that liberates fatty acids, an activity that PEDF can stimulate, suggesting a molecular pathway by which ligand/receptor interaction on the cell-surface could generate a cellular signal. Supported by the Intramural Research Program of the NIH, NEI.

Assignment of the patatin-like phospholipase domain containing 2 gene (PNPLA2) to porcine chromosome 2p17 with radiation hybrids

Assignment of the patatin-like phospholipase domain containing 2 gene (PNPLA2) to porcine chromosome 2p17 with radiation hybrids

Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU*

Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. University experimental research laboratory. Balb/c mice. First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.

Lysophospholipase A activity of Pseudomonas aeruginosa type III secretory toxin ExoU

Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU that is delivered directly into the eukaryotic cell via the type III secretion system. Recent studies demonstrated that ExoU has lipase activity, and that the cytotoxicity of ExoU is dependent on its patatin-like phospholipase domain. We investigated the phospholipase A (PLA) activity of ExoU. ExoU, but not non-catalytic ExoU- S142A, preincubated with the BEAS-2B cell lysate showed a weak increase of Ca 2+-independent PLA 2 activity. When activated ExoU was mixed with secretory type PLA 2, more phospholipase activity was observed, suggesting that ExoU has lysophospholipase A (lysoPLA) activity. A significant increase in lysoPLA activity was also observed. Glycerol enhanced this activity and inhibitors of iPLA 2 suppressed ExoU’s lysoPLA activity. Our results suggest that ExoU has a potent lysoPLA activity that requires the presence of the catalytically active site Ser 142 with an unknown eukaryotic cell factor(s) for its activation.