Group 1B phospholipase A2 in metabolic and inflammatory disease modulation

The group 1B phospholipase A₂ (PLA2G1B) is a secreted phospholipase that catalyzes the hydrolytic removal of the sn-2 fatty acyl moiety from phospholipids. This enzyme is synthesized most abundantly in the pancreas and is also expressed in the lung. The first part of this review article focuses on the role of pancreatic-derived PLA2G1B in mediating lipid absorption and discusses how the PLA2G1B-derived metabolic product contributes to cardiometabolic diseases, including obesity, hyperinsulinemia, hyperlipidemia, and atherosclerosis. The anti-helminth properties of PLA2G1B will also be discussed. The second part of this review will focus on PLA2G1B expressed in the lung, and in vitro data suggest that how this enzyme may modulate lung inflammation via both hydrolytic activity-dependent and -dependent mechanisms. Finally, recent studies revealing a relationship between PLA2G1B and cancer will also be discussed. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.     

Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2

We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism of the hVPLA(2)-induced LTB(4) biosynthesis in neutrophils, we thoroughly examined the effects of hVPLA(2) and their lipid products on the activity of group IVA cytosolic PLA(2) (cPLA(2)) and LTB(4) biosynthesis under different conditions. As low as 1 nm exogenous hVPLA(2) was able to induce the release of arachidonic acid (AA) and LTB(4). Typically, AA and LTB(4) were released in two phases, which were synchronized with a rise in intracellular calcium concentration ([Ca(2+)](i)) near the perinuclear region and cPLA(2) phosphorylation. A cellular PLA(2) assay showed that hVPLA(2) acted primarily on the outer plasma membrane, liberating fatty acids and lysophosphatidylcholine (lyso-PC), whereas cPLA(2) acted on the perinuclear membrane. Lyso-PC and polyunsaturated fatty acids including AA activated cPLA(2) and 5-lipoxygenase by increasing [Ca(2+)](i) and inducing cPLA(2) phosphorylation, which then led to LTB(4) biosynthesis. The delayed phase was triggered by the binding of secreted LTB(4) to the cell surface LTB(4) receptor, which resulted in a rise in [Ca(2+)](i) and cPLA(2) phosphorylation through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2. These results indicate that a main role of exogenous hVPLA(2) in neutrophil activation and LTB(4) biosynthesis is to activate cPLA(2) and 5-lipoxygenase primarily by liberating from the outer plasma membrane lyso-PC that induces [Ca(2+)](i) increase and cPLA(2) phosphorylation and that hVPLA(2)-induced LTB(4) production is augmented by the positive feedback activation of cPLA(2) by LTB(4).     

Dipalmitoylphosphatidylcholine (L-alpha-lecithin) stimulates phospholipase A2 activity in human amnion

In order to investigate the mechanism of dipalmitoylphosphatidylcholine (DPPC, L-alpha-lecithin) stimulation of the prostaglandin E (PGE) production of the amniotic membrane, effects of DPPC (50-800 micrograms/ml) on phospholipase A2 (PLA2), phospholipase C (PLC), PG endoperoxide synthase, and PGE synthase activities of human amniotic membrane were studied. Only PLA2 activity was increased by DPPC, suggesting that lecithin, the major surfactant component, increases the PGE production of the amniotic membrane by activating PLA2.     

Enzymatic properties of human cytosolic phospholipase A(2)gamma

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.     

Structure and mechanism of human cytosolic phospholipase A(2)

cPLA(2) is an 85-kDa enzyme whose primary function, the release of arachidonic acid from phospholipid membranes, is a crucial reaction in the metabolism of lipid mediators of inflammation. cPLA(2) consists of two domains: an N-terminal, C2-type unit analogous to those present in other membrane-targeting molecules, and a catalytic domain harboring an active site dyad at the bottom of a deep, mostly hydrophobic catalytic funnel. The absence of a third active site residue in the cPLA(2) cleft, as observed in other lipases, suggests that the enzyme proceeds through a novel catalytic mechanism. Crystallographic and biochemical studies of cPLA(2) will provide essential information for the development of small molecule inhibitors which may be employed in the control of inflammatory and other highly regulated processes.     

Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2). The purified recombinant protein has an apparent molecular weight of 16 kDa, identical to that of purified human placental or synovial fluid phospholipase A2, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Application of the purified protein onto several different gel filtration columns resulted in elution of the protein at molecular weights corresponding to 3.1-4.7 kDa, suggesting an interaction of the protein with the column resins. However, analytical ultracentrifugation experiments revealed that the protein behaves as a monomer (13.8-14.2 kDa) over a protein concentration range of approximately 10 micrograms/ml to 5 mg/ml. With autoclaved Escherichia coli membranes as substrate, the recombinant protein has catalytic properties (pH optimum, effects of bovine serum albumin, sodium chloride concentration, and requirement for calcium) similar to those of the protein purified from human placenta.     

Lipoprotein-associated phospholipase A2 (platelet-activating factor acetylhydrolase) and cardiovascular disease

PURPOSE OF REVIEW: Plasma lipoproteins carry a number of highly active enzymes in the circulation. One of these is lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), also known as platelet-activating factor acetylhydrolase. This review addresses the molecular properties of Lp-PLA(2), the controversy surrounding its role in atherosclerosis and the regulation of its plasma levels in humans. RECENT FINDINGS: Recent reports indicate that the enzyme Lp-PLA(2) found in both LDL and HDL may be independently regulated in these lipoprotein subclasses and have distinct roles in atherogenesis. Seminal findings establishing the response-to-retention hypothesis of atherosclerosis support further the potentially damaging role that in-situ release of LDL-associated oxidative products by Lp-PLA(2) may have in the formation of arterial wall lesions. In the mouse, where Lp-PLA(2) circulates mainly bound to HDL, overexpression leads to reduced atherosclerosis, raising the possibility that the enzyme in HDL may have a protective role. Further evidence for a potential protective role is seen in studies of partial or complete deficiency of the enzyme. In the more general setting of population studies, however, it is clear that Lp-PLA(2) is a positive risk factor for coronary disease and measurements of its mass may contribute to the prediction of coronary heart disease risk, especially in individuals with low LDL cholesterol levels. SUMMARY: Lp-PLA(2) is an enzyme with potentially multiple risks in atherosclerosis. In humans the weight of evidence suggests that it is a positive risk factor for coronary heart disease - an observation commensurate with its position in the direct pathological sequence leading from formation of oxidized LDL in the artery wall to cellular dysfunction and formation of lesions.     

Bimodal regulatory effect of melittin and phospholipase A2-activating protein on human type II secretory phospholipase A2

Melittin and phospholipase A2-activating protein (PLAP) are known as efficient activators of secretory phospholipase A2(sPLA2) types I, II, and III when phospholipid liposomes are used as substrate. The present study demonstrates that both peptides can either inhibit or activate sPLA2 depending on the peptide/phospholipid ratio when erythrocyte membranes serve as a biologically relevant substrate. Low concentrations of melittin and PLAP were observed to inhibit sPLA2-triggered release of fatty acids from erythrocyte membranes. The inhibition was reversed at melittin concentrations above 1 microM. PLAP-induced inhibition of sPLA2 persisted steadily throughout the used concentration range (0-150 nM). The two peptides induced a dose-dependent activation of sPLA2 at low concentrations, followed by inhibition when model membranes were used as substrate. This opposite modulatory effect on biological membranes and model membranes is discussed with respect to different mechanisms the interaction of the regulatory peptides with the enzyme molecules and the substrate vesicles.     

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.     

Group V phospholipase A(2)-mediated oleic acid mobilization in lipopolysaccharide-stimulated P388D(1) macrophages

P388D(1) macrophages prelabeled with [(3)H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A(2) and the group V secretory phospholipase A(2) (sPLA(2)). Here we show that when the LPS-activated cells are prelabeled with [(3)H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA(2) inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA(2) to the cells also triggers OA release. These data implicate a functionally active sPLA(2) as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A(2) is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D(1) macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.     

Detection of 14-kDa group II phospholipase A2 in human seminal plasma

About 90% of phospholipase A2 activity detected in human seminal plasma reacted with monoclonal antibodies raised against human synovial fluid phospholipase A2. The crude seminal plasma yielded a pure immuno-cross-reactive phospholipase A2 preparation in a single purification step using immuno-affinity chromatography. The amino acid sequence of the N-terminal 20 residues of this seminal enzyme was determined and found to be identical with that of human synovial phospholipase A2. Thus, it is suggested that human seminal plasma contains phospholipase A2, belonging to the 14-kDa group II enzyme family, as the major isoenzyme.     

Coordinate expression of secretory phospholipase A(2) and cyclooxygenase-2 in activated human keratinocytes

PGE₂ levels are altered in human epidermisafter in vivo wounding; however, mechanisms modulatingPGE₂ production in activated keratinocytes are unclear. Inprevious studies, we showed that PGE₂ is a growth-promotingautacoid in human primary keratinocyte cultures, and its production ismodulated by plating density, suggesting that regulatedPGE₂ synthesis is an important component of wound healing.Here, we examine the role of phospholipase A₂ (PLA₂) and cyclooxygenase (COX) enzymes in modulation ofPGE₂ production. We report that the increasedPGE₂ production that occurs in keratinocytes grown innonconfluent conditions is also observed after in vitro wounding,indicating that similar mechanisms are involved. This increase wasassociated with coordinate upregulation of both COX-2 and secretoryPLA₂ (sPLA₂) proteins. IncreasedsPLA₂ activity was also observed. By RT-PCR, we identifiedthe presence of type IIA and type V sPLA₂, along with theM-type sPLA₂ receptor. Thus the coordinate expression ofsPLA₂ and COX-2 may be responsible for the increasedprostaglandin synthesis in activated keratinocytes during wound repair.

     

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2)

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.     

Group IB phospholipase A2 from Pseudonaja textilis

Pseudonaja textilis, an Australian Elapid, is known to produce a highly toxic venom. Both protein profiling and N-terminal sequence analysis showed the presence of four new phospholipases A(2) in this venom. Besides being non-lethal, the phospholipase A(2) proteins were found to be moderately active enzymes and they showed procoagulant property. cDNA cloning and characterization indicated the presence of two isoforms of PLA(2) proteins in a single snake, each containing the "pancreatic loop," characteristic of group IB phospholipase A(2). The genomic cloning also confirmed the presence of two genes each containing four exons that are interrupted by three introns. Phylogenetic analysis showed that the venom group IB PLA(2) gene is primitive and could have evolved from the same ancestor as the mammalian and venom group IA PLA(2) genes. In the present study, we report that the Pt-PLA2 gene could be responsible for the production of PL1, 2, and 3 possibly via RNA editing process.     

Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells.

Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10(-5) M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes.