Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor

Summary The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the -amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.     

Simultaneous inhibition of anti‐coagulation and inflammation: crystal structure of phospholipase A2 complexed with indomethacin at 1.4 Å resolution reveals the presence of the new common ligand‐binding site

A novel ligand‐binding site with functional implications has been identified in phospholipase A₂ (PLA₂). The binding of non‐steroidal anti‐inflammatory agent indomethacin at this site blocks both catalytic and anti‐coagulant actions of PLA₂. A group IIA PLA₂ has been isolated from Daboia russelli pulchella (Russell's viper) which is enzymatically active as well as induces a strong anti‐coagulant action. The binding studies have shown that indomethacin reduces the effects of both anti‐coagulant and pro‐inflammatory actions of PLA₂. A group IIA PLA₂ was co‐crystallized with indomethacin and the structure of the complex has been determined at 1.4 Å resolution. The structure determination has revealed the presence of an indomethacin molecule in the structure of PLA₂ at a site which is distinct from the conventional substrate‐binding site. One of the carboxylic group oxygen atoms of indomethacin interacts with Asp 49 and His 48 through the catalytically important water molecule OW 18 while the second carboxylic oxygen atom forms an ionic interaction with the side chain of Lys 69. It is well known that the residues, His 48 and Asp 49 are essential for catalysis while Lys 69 is a part of the anti‐coagulant loop (residues, 54–77). Indomethacin binds in such a manner that it blocks the access to both, it works as a dual inhibitor for catalytic and anti‐coagulant actions of PLA₂. This new binding site in PLA₂ has been observed for the first time and indomethacin is the first compound that has been shown to bind at this novel site resulting in the prevention of anti‐coagulation and inflammation. Copyright © 2009 John Wiley & Sons, Ltd.     

Allosteric regulation by membranes and hydrophobic subsites in phospholipase A2 enzymes determine their substrate specificity

Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including obesity, insulin sensitivity, and cardiovascular diseases. However, defining the substrate specificity of enzymes of lipid metabolism is a challenging task. For example, phospholipase A₂ (PLA₂) enzymes constitute a superfamily of degradative, biosynthetic, and signaling enzymes that all act stereospecifically to hydrolyze and release the fatty acids of membrane phospholipids. This review focuses on how membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation and other diseases. Our group has developed “substrate lipidomics” to quantify the substrate phospholipid specificity of each PLA₂ and coupled this with molecular dynamics simulations to reveal that enzyme specificity is linked to specific hydrophobic binding subsites for membrane phospholipid substrates. We have also defined unexpected headgroup and acyl chain specificity for each of the major human PLA₂ enzymes, which explains the observed specificity at a structural level. Finally, we discovered that a unique hydrophobic binding site—and not each enzyme’s catalytic residues or polar headgroup binding site—predominantly determines enzyme specificity. We also discuss how PLA₂s release specific fatty acids after allosteric enzyme association with membranes and extraction of the phospholipid substrate, which can be blocked by stereospecific inhibitors. After decades of work, we can now correlate PLA₂ specificity and inhibition potency with molecular structure and physiological function.     

Structure/Function Relationships of Adipose Phospholipase A2 Containing a Cys-His-His Catalytic Triad

Adipose phospholipase A₂ (AdPLA or Group XVI PLA₂) plays an important role in the onset of obesity by suppressing adipose tissue lipolysis. As a consequence, AdPLA-deficient mice are resistant to obesity induced by a high fat diet or leptin deficiency. It has been proposed that AdPLA mediates its antilipolytic effects by catalyzing the release of arachidonic acid. Based on sequence homology, AdPLA is part of a small family of acyltransferases and phospholipases related to lecithin:retinol acyltransferase (LRAT). To better understand the enzymatic mechanism of AdPLA and LRAT-related proteins, we solved the crystal structure of AdPLA. Our model indicates that AdPLA bears structural similarity to proteins from the NlpC/P60 family of cysteine proteases, having its secondary structure elements configured in a circular permutation of the classic papain fold. Using both structural and biochemical evidence, we demonstrate that the enzymatic activity of AdPLA is mediated by a distinctive Cys-His-His catalytic triad and that the C-terminal transmembrane domain of AdPLA is required for the interfacial catalysis. Analysis of the enzymatic activity of AdPLA toward synthetic and natural substrates indicates that AdPLA displays PLA₁ in addition to PLA₂ activity. Thus, our results provide insight into the enzymatic mechanism and biochemical properties of AdPLA and LRAT-related proteins and lead us to propose an alternate mechanism for AdPLA in promoting adipose tissue lipolysis that is not contingent on the release of arachidonic acid and that is compatible with its combined PLA₁/A₂ activity.     

Structural characterization of myotoxic ecarpholin S from Echis carinatus venom

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. Ecarpholin S, isolated from the venom of the snake Echis carinatus sochureki, is a phospholipase A₂ (PLA₂) belonging to the Ser⁴⁹-PLA₂ subgroup. It has been characterized as having low enzymatic but potent myotoxic activities. The crystal structures of native ecarpholin S and its complexes with lauric acid, and its inhibitor suramin, were elucidated. This is the first report of the structure of a member of the Ser⁴⁹-PLA₂ subgroup. We also examined interactions of ecarpholin S with phosphatidylglycerol and lauric acid, using surface plasmon resonance, and of suramin with isothermal titration calorimetry. Most Ca²⁺-dependent PLA₂ enzymes have Asp in position 49, which plays a crucial role in Ca²⁺ binding. The three-dimensional structure of ecarpholin S reveals a unique conformation of the Ca²⁺-binding loop that is not favorable for Ca²⁺ coordination. Furthermore, the endogenously bound fatty acid (lauric acid) in the hydrophobic channel may also interrupt the catalytic cycle. These two observations may account for the low enzymatic activity of ecarpholin S, despite full retention of the catalytic machinery. These observations may also be applicable to other non-Asp⁴⁹-PLA₂ enzymes. The interaction of suramin in its complex with ecarpholin S is quite different from that reported for the Lys⁴⁹-PLA₂/suramin complex_, where the interfacial recognition face (i-face), C-terminal region, and N-terminal region of ecarpholin S play important roles. This study provides significant structural and functional insights into the myotoxic activity of ecarpholin S and, in general, of non-Asp⁴⁹-PLA₂ enzymes.     

Conservation of group XII phospholipase A2 from bacteria to human

Vertebrate group XII phospholipases A₂ (GXII PLA₂, conserved domain pfam06951) are proteins with unique structural and functional features within the secreted PLA₂ family. In humans, two genes (GXIIA PLA₂ and GXIIB PLA₂) have been characterised. GXIIA PLA₂ is enzymatically active whereas GXIIB PLA₂ is devoid of catalytic activity. Recently, putative homologues of the vertebrate GXII PLA₂s were described in non-vertebrates. In the current study a total of 170 GXII PLA₂ sequences were identified in vertebrates, invertebrates, non-metazoan eukaryotes, fungi and bacteria. GXIIB PLA₂ was found only in vertebrates and the searches failed to identify putative GXII PLA₂ homologues in Archaea. Comparisons of the predicted functional domains of GXII PLA₂s revealed considerable structural identity within the Ca^2 +-binding and the catalytic sites among the various organisms suggesting that functional conservation may have been retained across evolution. The preservation of GXII PLA₂ family members from bacteria to human indicates that they have emerged early in evolution and evolved via gene/genome duplication events prior to Eubacteria. Gene duplicates were identified in some invertebrate taxa suggesting that species-specific duplications occurred. The analysis of the GXII PLA₂ homologue genome environment revealed that gene synteny and gene order are preserved in vertebrates. Conservation of GXII PLA₂s indicates that important functional roles involved in species survival and were maintained across evolution and may be dependent on or independent of the enzyme's phospholipolytic activity.     

High-affinity selective inhibitor against phospholipase A2 (PLA2): a computational study

Phospholipase A₂ (PLA₂) is the most abundant protein found in snake venom. PLA₂ induces a variety of pharmacological effects such as neurotoxicity, myotoxicity and cardiotoxicity as well as anticoagulant, hemolytic, anti-platelet, hypertensive, hemorrhagic and edema inducing effects. In this study, the three dimensional structure of PLA₂ of Naja sputatrix (Malayan spitting cobra) was modeled by I-TASSER, SWISS-MODEL, PRIME and MODELLER programs. The best model was selected based on overall stereo-chemical quality. Further, molecular dynamics simulation was performed to know the stability of the modeled protein using Gromacs software. Average structure was generated during the simulation period of 10?ns. High throughput virtual screening was employed through different databases (Asinex, Hit finder, Maybridge, TOSLab and ZINC databases) against PLA₂. The top seven compounds were selected based on the docking score and free energy binding calculations. These compounds were analyzed by quantum polarized ligand docking, induced fit docking and density functional theory calculation. Furthermore, the stability of lead molecules in the active site of PLA₂ was employed by MD simulation. The results show that selected lead molecules were highly stable in the active site of PLA₂.     

Small-molecule inhibitors as potential therapeutics and as tools to understand the role of phospholipases A2

Phospholipase A₂ (PLA₂) enzymes are involved in various inflammatory pathological conditions including arthritis, cardiovascular and autoimmune diseases. The regulation of their catalytic activity is of high importance and a great effort has been devoted in developing synthetic inhibitors. We summarize the most important small-molecule synthetic PLA₂ inhibitors developed to target each one of the four major types of human PLA₂ (cytosolic cPLA₂, calcium-independent iPLA_2, secreted sPLA_2, and lipoprotein-associated LpPLA₂). We discuss recent applications of inhibitors to understand the role of each PLA₂ type and their therapeutic potential. Potent and selective PLA₂ inhibitors have been developed. Although some of them have been evaluated in clinical trials, none reached the market yet. Apart from their importance as potential medicinal agents, PLA₂ inhibitors are excellent tools to unveil the role that each PLA₂ type plays in cells and in vivo. Modern medicinal chemistry approaches are expected to generate improved PLA₂ inhibitors as new agents to treat inflammatory diseases.     

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A₂ (PLA₂). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA₂ toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA₂. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA₂. Moreover, it was found that PLA₂ and N-terminally mutated PLA₂ adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA₂ and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA₂, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA₂ at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA₂.     

X-ray crystal structure and molecular dynamics simulation of bovine pancreas phospholipase A2–n-dodecylphosphorylcholine complex

The crystal structure of n-dodecylphosphorylcholine (n-C₁₂PC)–bovine pancreas phospholipase A₂ (PLA₂) complex provided the following structural.characteristics: (1) the dodecyl chain of n-C₁₂PC was located at the PLA₂ N -terminal helical region by hydrophobic interactions, which corresponds to the binding pocket of 2-acyl fatty acid chain (β-chain) of the substrate phospholipid, (2) the region from Lys-53 to Lys-56 creates a cholinereceiving pocket of n-C₁₂PC and (3) the N-termillal group of Ala-1 shifts significantly toward the Tyr-52 OH group by the binding of the n-C1₂PC inhibitor. Since the accuracy of the X-ray analysis (R = 0.275 at 2.3 Å resolution) was insufficient to establish these important X-ray insights, the complex structure was further investigated through the molecular dynamics (M D) simulation, assuming a system in aqueous solution at 310K. The M D simulation covering 176 ps showed that the structural characteristics observed by X-ray analysis are intrinsic and also stable in the dynamic state. Furthermore, the M D simulation made clear that the PLA₂ binding pocket is large enough to permit the conformational fluctuation of the n-C₁₂PC hydrocarbon chain. © 1994 Wiley-Liss, Inc. © 1994 Wiley-Liss, Inc.     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A₂ class

Phospholipases A₂ (PLA₂s) are enzymes responsible for membrane disruption through Ca²⁺‐dependent hydrolysis of phospholipids. Lys49‐PLA₂s are well‐characterized homologue PLA₂s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA₂s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49‐PLA₂ (BthTX‐II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49‐PLA₂ conserves all the residues responsible for Ca²⁺ coordination and of the catalytic network, features thought to be fundamental for PLA₂ enzymatic activity. Previous crystallographic studies of apo BthTX‐II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX‐II is not catalytic based on an in vitro cell viability assay and time‐lapse experiments on C2C12 myotube cell cultures, X‐ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX‐II is devoid of catalytic activity, as already observed for Lys49‐PLA₂s. Crystallographic studies of the complex BthTX‐II/Ca²⁺ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca²⁺ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX‐II is more phylogenetically related to Lys49‐PLA₂s than to other Asp49‐PLA₂s, thus allowing Crotalinae subfamily PLA₂s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Computational and in vitro insights on snake venom phospholipase A2 inhibitor of phytocompound ikshusterol3-O-glucoside of Clematis gouriana Roxb. ex DC.

Ikshusterol3-O-glucoside was isolated from Clematis gouriana Roxb. ex DC. root. A structure of the isolated compound was determined on the basis of various spectroscopic interpretations (UV, NMR, FTIR, and GC-MS-EI). This structure was submitted in the PubChem compound database (SID 249494133). SID 249494133 was carried out by density functional theory calculation to observe the chemical stability and electrostatic potential of this compound. The absorption, distribution, metabolism, and excretion property of this compound was predicted to evaluate the drug likeness and toxicity. In addition, molecular docking, quantum polarized ligand docking, prime MMGBSA calculation, and induced fit docking were performed to predict the binding status of SID 249494133 with the active site of phospholipase A₂ (PLA₂) (PDB ID: 1A3D). The stability of the compound in the active site of PLA₂ was carried out using molecular dynamics simulation. Further, the anti-venom activity of the compound was assessed using the PLA₂ assay against Naja naja (Indian cobra) crude venom. The results strongly show that Ikshusterol3-O-glucoside has a potent snake-venom neutralizing capacity and it might be a potential molecule for the therapeutic treatment for snakebites.     

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin

The myotoxic mechanism for PLA₂-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA₂-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA₂-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA₂-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA₂-like toxins in general.     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

Molecular conformation of prostaglandin E2

The crystal and molecular structure of prostaglandin E₂ (PGE₂) has been determined by X-ray diffraction. The compound crystallizes in the triclinic space group P1 with Z = 1 and , , , α = 87.347°, β = 94.042°, and γ = 91.010°. Gauche-gauche interactions appear in both side chains. The efficient molecular packing and hydrogen bonding network appears to stabilize the observed molecular conformation.     

Virtual analysis of structurally diverse synthetic analogs as inhibitors of snake venom secretory phospholipase A2

Due to the toxic pathophysiological role of snake venom phospholipase A₂ (PLA₂), its compelling limitations to anti‐venom therapy in humans and the need for alternative therapy foster considerable pharmacological interest towards search of PLA₂ specific inhibitors. In this study, an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on VRV‐PL‐V (Vipera russellii venom phospholipase A₂ fraction—V) belonging to Group II‐B secretory PLA₂ from Daboia russelli pulchella is carried out in order to study the structure‐based inhibitor design. The accuracy of the model was validated using multiple computational approaches. The molecular docking study of this protein was undertaken using different classes of experimentally proven, structurally diverse synthetic inhibitors of secretory PLA₂ whose selection is based on IC₅₀ value that ranges from 25 μM to 100 μM. Estimation of protein–ligand contacts by docking analysis sheds light on the importance of His 47 and Asp 48 within the VRV‐PL‐V binding pocket as key residue for hydrogen bond interaction with ligands. Our virtual analysis revealed that compounds with different scaffold binds to the same active site region. ADME analysis was also further performed to filter and identify the best potential specific inhibitor against VRV‐PL‐V. Additionally, the e‐pharmacophore was generated for the best potential specific inhibitor against VRV‐PL‐V and reported here. The present study should therefore play a guiding role in the experimental design of VRV‐PL‐V inhibitors that may provide better therapeutic molecular models for PLA₂ recognition and anti‐ophidian activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A₂ (PLA₂) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA₂ and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA₂ and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA₂. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA₂ and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA₂ activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca²⁺ binding and antigenicity of Lys-6-modified PLA₂ were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA₂ to 32% of that of PLA₂. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA₂ activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA₂ is almost the same as that of native PLA₂. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA₂, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA₂.     

Phospholipases A2 in Ischemic and Toxic Brain Injury

Phospholipases A₂ (PLA₂s) regulate hydrolysis of fatty acids, including arachidonic acid, from the sn-2 position of phospholipid membranes. PLA₂ activity has been implicated in neurotoxicity and neurodegenerative processes secondary to ischemia and reperfusion and other oxidative stresses. The PLA₂s constitute a superfamily whose members have diverse functions and patterns of expression. A large number of PLA₂s have been identified within the central nervous systems of rodents and humans. We postulated that group IV large molecular weight, cytosolic phospholipase A₂ (cPLA₂) has a unique role in neurotoxicity associated with ischemic or toxin stress. We created mice deficient in cPLA₂ and tested this hypothesis in two injury models, ischemia/reperfusion and MPTP neurotoxicity. In each model cPLA₂ deficient mice are protected against neuronal injury when compared to their wild type littermate controls. These experiments support the hypothesis that cPLA₂ is an important mediator of ischemic and oxidative injuries in the brain.     

Arachidonic acid release from NIH 3T3 cells by group-I phospholipase A2: Involvement of a receptor-mediated mechanism

Group I pancreatic phospholipase A₂ (PLA₂ I) is primarily a digestive enzyme. Recently, however, in addition to its catalytic activity a receptor-mediated function has been described for this enzyme. PLA₂ I binding to its receptor induces cellular chemokinesis, proliferation, and smooth muscle contraction. This enzyme also induces the production of prostaglandin E₂ in certain cells and may have a proinflammatory role. However, despite its ability to hydrolyze phospholipids in in vitro assays, PLA₂-I does not efficiently catalyze release of AA from intact cells. Here, we demonstrate that while short-term exposure of NIH 3T3 cells to PLA₂-I is ineffective, exposure of 6 h or longer significantly increases the basal release of AA. Dose-response curve of PLA₂-I-induced AA release was saturable with an EC₅₀ of 14.01 ± 1.36 nM (n = 3). [³H]-AA was preferentially released over [³H]-oleic acid by PLA₂-I, inactivated with 4-bromophenacyl bromide, was fully capable of mediating AA release. These data suggest that a non-catalytic, receptor-mediated mechanism is involved in PLA₂-I-induced AA release in NIH-3T3 cells. This relase of AA is not dependent on protein kinase C or Ca²⁺ concentration. Comparison of the effect of PLA₂-I with those of ATP and platelet-derived growth factor indicates that each of these agonists regulates AA release via independent pathways. Neither the basal enzymatic activity of the 85-kDa cytosolic PLA₂ nor the protein level of this enzyme was affected by treatment of cells with PLA₂-I. However, the increase in basal enzymatic activity of 85 kDa PLA₂ due to protein kinase C activation was further enhanced by pretreatment of cells with PLA₂-I. We conclude that: (1) short-term exposure of cells to PLA₂ I does not cause measurable AA release; (2) release of AA from intact cells by this enzyme requires long-term exposure; (3) AA release is not mediated by a direct catalytic effect of PLA₂ I; and (4) AA release by PLA₂ I is accomplished via a receptor-mediated process. Taken together, these results raise the possibility that PLA₂ I, in addition to its digestive function, may also contribute to aggravate preexisting inflammatory processes and/or to initiate new ones when chronic exposure of cells to this enzyme occurs. © 1995 Wiley-Liss Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.