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Group IIA secreted phospholipase A 2 (group IIA sPLA 2 ) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of native stingray and dromedary groups V, IIA, and IB sPLA 2 s on several Gram-positive and Gram-negative strains. The rank order potency among both marine and mammal sPLA 2 s against Gram-positive bacteria is group IIA > V > IB, whereas Gram-negative bacteria exhibited a much higher resistance. There is a synergic action of the sPLA 2 with lysozyme when added to the bacteria culture prior to sPLA 2 .The bactericidal efficiency of groups V and IIA sPLA 2 s was shown to be dependent upon the presence of calcium ions and to a less extent Mg 2+ ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray and dromedary groups V, IIA, and IB sPLA 2 s present no cytotoxicity after their incubation with MDA-MB-231cells. stingray groups V and IIA sPLA 2 s, like mammal ones, may be considered as future therapeutic agents against bacterial infections.

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.

Biologically active F‐ and E/D‐type‐prostane ring isomers (F 2 ‐IP and E 2 /D 2 ‐IP, respectively) are produced in situ by non‐enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho‐IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet‐activating factor acetylhydrolases (PAF‐AH) are the main endogenous PLA 2 involved in degradation of PtdCho‐IP. The present study shows that the PtdCho‐IP are also subject to hydrolysis by group IIA, V and X secretory PLA 2 , which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho‐IP of auto‐oxidized plasma lipoproteins after incubation for 1–4 h (37 °C) in the absence or presence of recombinant human sPLA 2 (1–2.5 µg/ml). In the absence of exogenously added sPLA 2 the total PtdCho‐IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL 3 , respectively. In the presence of group V or group X sPLA 2 (2.5 µg/ml), the PtdCho‐IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA 2 (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8–24 h incubation. This report provides the first demonstration that PtdCho‐IP are readily hydrolyzed by group IIA, V and X sPLA 2 . A co‐location of sPLA 2 and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.

We examined the functional role of 14-kD secretory group V phospholipase A(2) (gVPLA(2)) on the barrier function of pulmonary endothelial cells (ECs) after LPS activation in vitro. Expression of gVPLA(2) was elicited by 20 ng/ml LPS as demonstrated by increased (1) mRNA, (2) protein content, and (3) cell surface expression of gVPLA(2) within 4 hours. The effect of LPS on EC barrier function was measured by transendothelial monolayer electrical resistance (TER). LPS increased permeability across EC monolayers at 2-3 hours, and was sustained for 10 hours or more. Blockade of gVPLA(2) with mouse monoclonal 3G1 (MCL-3G1) monoclonal antibody directed against gVPLA(2) inhibited EC barrier dysfunction elicited by LPS in a time- and concentration-dependent manner; control IgG had no effect on TER. Like LPS, exogenous gVPLA(2) caused increased EC permeability in a time- and concentration-dependent manner; neither gIIaPLA(2), a close homolog of gVPLA(2), nor W31A, an inactive mutant of gVPLA(2), caused a decrease in EC TER. Immunofluorescence analysis revealed comparable F-actin stress fiber and intercellular gap formation for ECs treated with either gVPLA(2) or LPS. Treatment with gVPLA(2) disrupted vascular endothelial-cadherin junctional complexes on ECs. Coincubation of ECs with MCL-3G1 substantially attenuated the structural changes caused by gVPLA(2) or LPS. We demonstrate that (1) gVPLA(2) is constitutively expressed in ECs and is up-regulated after LPS activation, (2) endogenously secreted gVPLA(2) from ECs after LPS increases EC permeability through F-actin and junctional complex rearrangement, and (3) inhibition of endogenous gVPLA(2) from ECs is sufficient to block disruption of the EC barrier function after LPS in vitro.

Secretory phospholipases A 2 (sPLA 2 s) are a diverse family of low molecular mass enzymes (13–18 kDa) that hydrolyze the sn -2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA 2 (sPLA 2 -X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA 2 (sPLA 2 -V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA 2 -X in several respects. Although sPLA 2 -V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA 2 -X. In addition, the requirement of Ca 2+ for the lipolysis of LDL was about 10-fold higher for sPLA 2 -V than sPLA 2 -X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA 2 -V in the presence of sodium citrate, which contrasted with the potent response to sPLA 2 -X. Moreover, sPLA 2 -X, but not sPLA 2 -V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA 2 -X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K d = 3.1 nM) in the presence of Ca 2+ . Selective interaction of sPLA 2 -X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.