A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains.

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

The Cytosolic Phospholipase A2α N-terminal C2 Domain Binds and Oligomerizes on Membranes with Positive Curvature.

Group IV phospholipase A2α (cPLA2α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA2α to the Golgi involves the N-terminal C2 domain, whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA2α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigated how membrane curvature influences the homeostatic role of cPLA2α in vesicular trafficking. The cPLA2α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA2α membrane penetration. We showed that cPLA2α undergoes C2 domain-dependent oligomerization on membranes in vitro and in cells. We found that the association of the cPLA2α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization was observed on vesicles ~50 nm in diameter. We demonstrated that the cPLA2α C2 domain localizes to cholesterol enriched Golgi-derived vesicles independently of cPLA2α catalytic activity. Moreover, we demonstrate the C2 domain selectively localizes to lipid droplets whereas the full-length enzyme to a much lesser extent. Our results therefore provide novel insight into the molecular forces that mediate C2 domain-dependent membrane localization in vitro and in cells.

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Structural basis of phosphatidylcholine recognition by the C2-domain of cytosolic phospholipase A2α.

Ca2+-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLA2α activity. The DHPC-binding mode of the cPLA2α C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains.

The role of intracellular anionic phospholipids in the production of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ɛ.

N-Acyl-phosphatidylethanolamines (NAPEs) represent a class of glycerophospholipids and serve as the precursors of bioactive N-acylethanolamines, including arachidonoylethanolamide (anandamide), palmitoylethanolamide and oleoylethanolamide. NAPEs are produced in mammals by N-acyltransferases, the enzymes which transfer an acyl chain of glycerophospholipids to the amino group of phosphatidylethanolamine. Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ, also called PLA2G4E) was identified as Ca2+-dependent N-acyltransferase. We showed that the activity is remarkably stimulated by phosphatidylserine (PS) in vitro. In the present study, we investigated whether or not endogenous PS regulates the function of cPLA2ɛ in living cells. When PS synthesis was suppressed by the knockdown of PS synthases in cPLA2ɛ-expressing cells, the cPLA2ɛ level and its N-acyltransferase activity were significantly reduced. Mutagenesis studies revealed that all of C2, lipase and polybasic domains of cPLA2ɛ were required for its proper localization as well as the enzyme activity. Liposome-based assays showed that several anionic glycerophospholipids, including PS, phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, enhance the Ca2+-dependent binding of purified cPLA2ɛ to liposome membrane and stimulate its N-acyltransferase activity. Altogether, these results suggested that endogenous PS and other anionic phospholipids affect the localization and enzyme activity of cPLA2ɛ.

Associations of maternal PLA2G4C and PLA2G4D polymorphisms with the risk of spontaneous preterm birth in a Chinese population.

Preterm birth is the leading cause of mortality and morbidity in infants. Its etiology is multifactorial with genes and immune homeostasis. The authors investigated whether prostaglandin (PG) synthesis related single nucleotide polymorphisms (SNPs) PLA2G4C rs1366442 and PLA2G4D rs4924618 were associated with the risk of spontaneous preterm birth (SPTB) in a Chinese population of 114 cases of SPTB and 250 controls of term delivery. The risk associations were determined by odds ratios (ORs) and their 95% confidence intervals (CIs) calculated using multivariate logistic regression. Homology modeling was performed to elucidate potential mechanism of the SNP function. The maternal AT/TT genotype of PLA2G4D rs4924618 was associated with a reduced risk of SPTB (OR, 0.61; 95% CI, 0.37­0.99), while no significant association between PLA2G4C rs1366442 and SPTB risk was identified. Structure and sequence analysis revealed that the amino acid substitution introduced by this SNP located at the conserved central core of the catalytic domain of cytosolic phospholipase A2 δ and was close to the active site. These findings suggested that the polymorphism of PLA2G4D rs4924618 may have a protective influence on the SPTB susceptibility in a Chinese population, supporting a role for genetics in the association between PG synthesis and preterm birth.

1-Heteroaryl-3-phenoxypropan-2-ones as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: Effect of the replacement of the ether oxygen with sulfur and nitrogen moieties on enzyme inhibition and metabolic stability.

Cytosolic phospholipase A2α (cPLA2α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgesic and anti-inflammatory drugs. We recently reported that certain 3-phenoxy-substituted 1-heteroarylpropan-2-ones are inhibitors of cPLA2α and/or FAAH. Starting from 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylic acid (3) and 1-(1H-benzotriazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (4), the effect of the replacement of the oxygen in position 3 of the propan-2-one scaffold by sulfur and nitrogen containing moieties on inhibition of cPLA2α and fatty acid amide hydrolase as well as on metabolic stability in rat liver S9 fractions was investigated. As a result of these structure-activity relationship studies it was found that the ether oxygen is of great importance for enzyme inhibitory potency. Replacement by sulfur led to an about 100-fold decrease of enzyme inhibition, nitrogen and substituted nitrogen atoms at this position even resulted in inactivity of the compounds. The effect of the structural variations performed on metabolic stability of the important ketone pharmacophore was partly different in the two series of compounds. While introduction of SO and SO2 significantly increased stability of the ketone against reduction in case of the indole-5-carboxylic acid 3, it had no effect in case of the benzotriazole 4. Further analysis of the metabolism of 3 and 4 in rat liver S9 fractions revealed that the major metabolite of 3 was the alcohol 53 formed by reduction of the keto group. In contrast, in case of 4 beside keto reduction an excessive hydroxylation of the terminal phenoxy group occurred leading to the dihydroxy compound 50. Experiments with enzyme inhibitors showed that the phenylhydroxylation of 4 was catalyzed by tranylcypromine sensitive cytochrome P450 isoforms, while the reduction of the ketone function of 3 and 4 was mainly caused by cytosolic short chain dehydrogenases/reductases (cSDR).

Selectivity of cytosolic phospholipase A2 type IV toward arachidonyl phospholipids.

Cytosolic phospholipase A2 (cPLA2 ) is an interesting protein involved in inflammatory processes and various diseases. Its catalytic mechanism as well as its substrate specificity for arachidonyl phospholipids is not typical for other phospolipases. Furthermore, a lid structure, which ensures a hydrophilic surface of the protein without any substrate bound and the movement of this flexible loop to make the hydrophobic active site accessible, is of high interest. Therefore, the focus of this work was to determine the binding mode of cPLA2 with various substrates, such as arachidonic acid, a synthetic inhibitor, a saturated phospholipid, and most importantly an arachidonyl phospholipid. To understand the selectivity of the protein toward the arachidonyl phospholipid and the interaction in a protein-ligand complex, molecular dynamics simulations were performed using the GROMOS suite of simulation programs. The simulations provide insight into the protein and showed that selective binding of arachidonyl phospholipids is because of the shape of the sn-2 tail. The amino acids Asn555 and Ala578 are involved in the strongest interactions observed in the protein-ligand complexes.

The development of an ELISA for group IVA phospholipase A2 in human red blood cells.

An immunoassay for IVA phospholipase A2 in human red blood cells is described. The assay is a non-competitive sandwich assay in which increasing amounts of the measured protein produce increased luminescence. The antibodies used in the assay are directed against two unique epitopes of the molecule, which sequentially trap and detect the protein. The standard curve covers the range 0.7ng to 23ng/mL (0.07 to 2.3ng/well). The intra-assay and inter-assay coefficients of variation were 9% and 12%, respectively. Evidence is presented that the assay is specific for the alpha paralog of IV PLA2. The assay allows simple and rapid quantification of IVAPLA2 in red blood cell lysates and other biological fluids.

Cytosolic phospholipase A2ε drives recycling through the clathrin-independent endocytic route.

Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.

Probing selected structural regions in the secreted phospholipase A2 from Arabidopsis thaliana for their impact on stability and activity.

In contrast to the well characterized secreted phospholipases A2 (sPLA2) from animals, their homologues from plants have been less explored. Their production in purified form is more difficult, and no data on their stability are known. In the present paper, different variants of the sPLA2 isoform α from Arabidopsis thaliana (AtPLA2α) were designed using a new homology model with the aim to probe the impact of regions that are assumed to be important for stability and catalysis. Moreover tryptophan residues were introduced in critical regions to enable stability studies by fluorescence spectroscopy. The variants were expressed in Escherichia coli and the purified enzymes were analyzed to get first insights into the peculiarities of structure stability and structure activity relationships in plant sPLA2s in comparison with the well-characterized homologous enzymes from bee venom and porcine pancreas. Stability data of the AtPLA2 variants obtained by fluorescence or CD measurements of the reversible unfolding by guanidine hydrochloride and urea showed that all enzyme variants are less stable than the enzymes from animal sources although a similar tertiary core structure can be assumed based on molecular modeling. More extended loop structures at the N-terminus in AtPLA2α are suggested to be the main reasons for the much lower thermodynamic stabilities and cooperativities of the transition curves. Modifications in the N-terminal region (insertion, deletion, substitution by a Trp residue) exhibited a strong positive effect on activity whereas amino acid exchanges in other regions of the protein such as the Ca(2+)-binding loop and the loop connecting the two central helices were deleterious with respect to activity.

Citrus allergy from pollen to clinical symptoms.

Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and ß) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.

The molecular basis of ceramide-1-phosphate recognition by C2 domains.

Group IVA cytosolic phospholipase A2 (cPLA2α), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affinity for a cationic patch in the C2 domain ß-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA2α-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specificity is mediated by Arg59, Arg6¹, and His6² (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confirm the origin of C1P specificity, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the first study demonstrating the molecular origin of C1P recognition.

C2-di-ethyl-ceramide-1-phosphate as an inhibitor of group IVA cytosolic phospholipase A2.

Ceramide-1-phosphate (C1P) has been shown to bind with C2 domain in group IVA cytosolic phospholipase A(2) (cPLA(2)α, PLA2G4A) and activate the enzyme activity directly. In cells, C1P causes translocation of cPLA(2)α to perinuclear regions including the Golgi complex by interacting with C2 domain in the enzyme, and then cPLA(2)α releases arachidonic acid from substrate phospholipids in the regions. In this study, we synthesized new di-ethyl (DE) phosphate ester analogs of C1P with N-acyl chains of different lengths, and examined their effects on cPLA(2)α. A DE-C1P analog with a C2-N-acyl chain (C2-DE-C1P), but not DE-C1P analogs with longer N-acyl chain, such as C6- and C16-DE-C1P, inhibited release of arachidonic acid via cPLA(2)α activation in CHO-W11A cells expressing platelet-activating factor (PAF) receptors without changing secretory phospholipase A(2)-induced release. Treatment with C2-DE-C1P did not modify phosphorylation of extracellular signal-regulated kinase 1/2 and cPLA(2)α and increase of intracellular Ca(2+) level induced by PAF, but inhibited Ca(2+)- and PAF-induced accumulation of cPLA(2)α in the Golgi complex. Phosphatidylcholine vesicles containing C2-DE-C1P reduced cPLA(2)α activity in vitro. C2-DE-C1P disturbed the binding of the enzyme to glycerophospholipids in the lipid-protein overlay assay, and the reagent alone did not bind to the enzyme. Interestingly, C2-DE-C1P inhibited neither Ca(2+)- and PAF-induced accumulation of C2 domain of cPLA(2)α in the Golgi complex nor binding of cPLA(2)α to C16-C1P. These results suggest that C2-DE-C1P appeared to inhibit cPLA(2)α, probably by interaction with a site in the catalytic domain of the enzyme, not with the site in C2 domain responsible for native C1P.

C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes.

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA(2)α binds zwitterionic lipids such as phosphatidylcholine in a Ca(2+)-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA(2)α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA(2)α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA(2)α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA(2)α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA(2)α.

Binding conformation of 2-oxoamide inhibitors to group IVA cytosolic phospholipase A2 determined by molecular docking combined with molecular dynamics.

The group IVA cytosolic phospholipase A(2) (GIVA cPLA(2)) plays a central role in inflammation. Long chain 2-oxoamides constitute a class of potent GIVA cPLA(2) inhibitors that exhibit potent in vivo anti-inflammatory and analgesic activity. We have now gained insight into the binding of 2-oxoamide inhibitors in the GIVA cPLA(2) active site through a combination of molecular docking calculations and molecular dynamics simulations. Recently, the location of the 2-oxoamide inhibitor AX007 within the active site of the GIVA cPLA(2) was determined using a combination of deuterium exchange mass spectrometry followed by molecular dynamics simulations. After the optimization of the AX007-GIVA cPLA(2) complex using the docking algorithm Surflex-Dock, a series of additional 2-oxoamide inhibitors have been docked in the enzyme active site. The calculated binding affinity presents a good statistical correlation with the experimental inhibitory activity (r(2) = 0.76, N = 11). A molecular dynamics simulation of the docking complex of the most active compound has revealed persistent interactions of the inhibitor with the enzyme active site and proves the stability of the docking complex and the validity of the binding suggested by the docking calculations. The combination of molecular docking calculations and molecular dynamics simulations is useful in defining the binding of small-molecule inhibitors and provides a valuable tool for the design of new compounds with improved inhibitory activity against GIVA cPLA(2).

Structural and functional characterization of a γ-type phospholipase A2 inhibitor from bothrops jararacussu snake plasma.

Phospholipases A2 (PLA2s) from snake venoms comprise a group of 14-18 kDa proteins, responsible for several toxic effects induced by the whole venom. Considering this, studies aiming at the search for natural inhibitors of these proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a γ-type phospholipase A2 inhibitor (PLI) from Bothrops jararacussu snake plasma, named γBjussuMIP. This acidic glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-Sepharose 4B coupled with BthTXII, showing a pI ∼ 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on Sephacryl S-200). The interaction between γBjussuMIP (MIP) and Phospholipase A2 (PLA2) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1:1 molar mixture was higher than that calculated for the addition of the spectra of the unbound proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA2 participate in proteins interaction as blue shift of 4 nm was observed. The γBjussuMIP cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature protein of 181 amino acid residues. Alignment of its amino acid sequence with those of other snake γPLIs showed 89-94% of similarity. γBjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA2s, such as phospholipase, anticoagulant, myotoxic, edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by γBjussuMIP.

Phospholipase A2 inhibitors (ßPLIs) are encoded in the venom glands of Lachesis muta (Crotalinae, Viperidae) snakes.

Phospholipase A(2) inhibitors (PLIs) are glycoproteins secreted by snake liver into the circulating blood aiming the self-protection against toxic venom phospholipases A(2). In the present study, we describe the first complete nucleotide sequence of a ßPLI from venom glands of a New World snake, Lachesis muta. The deduced primary structure was compared to other known ßPLIs and recent literature findings of other possible roles of PLIs in snakes are discussed.

Interfacial kinetic and binding properties of mammalian group IVB phospholipase A2 (cPLA2beta) and comparison with the other cPLA2 isoforms.

The cytosolic (group IV) phospholipase A(2) (cPLA(2)s) family contains six members. We have prepared recombinant proteins for human α, mouse ß, human γ, human δ, human ε, and mouse ζ cPLA(2)s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA(2)ß action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca(2+) only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca(2+) on the interfacial binding of mouse cPLA(2)ß and its C2 domain to vesicles. Ca(2+)-dependent binding of mouse cPLA(2)ß to cardiolipin-containing vesicles requires a patch of basic residues near the Ca(2+)-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA(2)δ also displays Ca(2+)- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A(1), and phospholipase A(2) activities of the full set of mammalian cPLA(2)s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA(2)δ stands out as having relatively high phospholipase A(1) activity. We also tested the susceptibility of all cPLA(2) family members to a panel of previously reported inhibitors of human cPLA(2)α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA(2)ß. These in vitro studies help determine the regulation and function of the cPLA(2) family members.

Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation.

α-type phospholipase A2 inhibitory protein (PLIα) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIα, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIα-LP, is homologous to GbPLIα, but it does not show any inhibitory activity against PLA2s. When a mixture of denaturant-treated monomeric forms of GbPLIα and EqPLIα-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIα and EqPLIα-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIα and the recombinant chimeric EqPLIα-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLIα, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIα-LP and the recombinant chimeric GbPLIα, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLIα-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIα were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIα with a single amino acid substitution, GbPLIα(K28E).