Phospholipase A2 from sheep erythrocyte membrane CA2+ dependence and localization

The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A₂ specificity was found to be responsible for both phosphatidylethanolamine and phosphatidylcholine degradation.The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [¹⁴C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.     

Probing Calcium Ion-Induced Conformational Changes of Taiwan Cobra Phospholipase A2 by Trinitrophenylation of Lysine Residues

Phospholipase A₂ (PLA₂) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca²⁺. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca²⁺. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca²⁺ when Ca²⁺ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca²⁺. Alternatively, the nonessentiality of Lys-65 for PLA₂ activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca²⁺, suggesting that Ca²⁺ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca²⁺ are well consistent with their functional roles in the catalytic mechanism of PLA₂, and suggest that the occurrence of conformational changes with PLA₂ could be explored by chemical modification studies.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

1-Cysteine peroxiredoxin: A dual-function enzyme with peroxidase and acidic Ca-independent phospholipase A2 activities

Peroxiredoxins (Prx) are enzymes that catalyze the reduction of hydrogen peroxide and alkyl hydroperoxides. Prxs are ubiquitous enzymes with representatives found in Bacteria, Archaea and Eukarya. Many 1-cysteine peroxiredoxins (1-CysPrx) are dual-function enzyme with both peroxidase and acidic Ca²⁺-independent phospholipase A₂ (aiPLA₂) activities. The functions proposed for 1-CysPrx/aiPLA₂ include the protection of cell membrane phospholipids against oxidative damage (peroxidation) and the metabolism (hydrolysis) of phospholipids, such as those of lung surfactant. The peroxidase active site motif PVCTTE of 1-CysPrx contains the conserved catalytic cysteine residue, and the esterase (lipase) motif GXSXG of the enzyme contains the conserved catalytic serine residue. In addition to the classic lipase motif GXSXG, various 1-CysPrx/aiPLA₂s have closely related variant putative lipase motifs containing the catalytic serine residue. The PLA₂ moieties are prevalent and highly homologous in vertebrate and bacterial 1-CysPrx/aiPLA₂s that is consistent with a high degree evolutional conservation of the enzyme.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

Phospholipase A2 and arachidonic acid in Alzheimer's disease

Essential fatty acids (EFA) play a critical role in the brain and regulate many of the processes altered in Alzheimer's disease (AD). Technical advances are allowing for the dissection of complex lipid pathways in normal and diseased states. Arachidonic acid (AA) and specific isoforms of phospholipase A₂ (PLA₂) appear to be critical mediators in amyloid-β (Aβ)-induced pathogenesis, leading to learning, memory, and behavioral impairments in mouse models of AD. These findings and ongoing research into lipid biology in AD and related disorders promise to reveal new pharmacological targets that may lead to better treatments for these devastating conditions.     

Phospholipase A2-derived lysophosphatidylcholine triggers Ca entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca²⁺ entry through sarcolemmal cationic channels. Using the Mn²⁺ quench method, we show here that Mn²⁺ entry triggered by Ca²⁺ store depletion but not basal Mn²⁺ entry relies on Ca²⁺-independent PLA₂ (iPLA₂) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx^5cv mouse. iPLA₂ was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA₂ lipid product lysophosphatidylcholine was found to trigger Ca²⁺ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA₂ and lysophospholipid production may be of interest to reduce Ca²⁺ entry and subsequent degeneration of dystrophic muscle.     

Shape relaxations in a fluid supported membrane during hydrolysis by phospholipase A2

The behavior of a fluid supported membrane during hydrolysis by phospholipase A₂ is for the first time visualized by time-resolved fluorescence imaging. After a lag phase, hydrolysis proceeds from the boundary of existing holes and via nucleation of new holes. During subsequent hydrolysis, the shape of the membrane boundary is determined both by hydrolysis and by shape relaxations due to the action of line tension. This is manifested by the appearance of Rayleigh instabilities in membrane rims and by an effect analogous to domain coarsening in phase transitions in which membrane holes decay when they are within a certain distance from larger and expanding holes.     

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host

Type-IIA secreted phospholipase A₂ (sPLA₂-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA₂-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA₂-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA₂-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA₂-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA₂-IIA. Transgenic mice over-expressing human sPLA₂-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA₂-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA₂-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA₂-IIA, indicating that instilled sPLA₂-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA₂-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Cytoplasmic phospholipase A2 antagonists inhibit multiple endocytic membrane trafficking pathways

Previous studies have suggested a role for cytosolic Ca²⁺-independent phospholipase A₂ (PLA₂) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA₂ requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA₂ antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA₂ plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules.     

Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

Among the emerging phospholipase A₂ (PLA₂) superfamily, the secreted PLA₂ (sPLA₂) family consists of low-molecular-mass, Ca²⁺-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA₂ enzymes have been identified in mammals. Individual sPLA₂s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA₂ enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA₂ isoforms fueled by studies employing transgenic and knockout mice for several sPLA₂s.     

Insights on the structure of native CNF,an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake

Several snake species possess endogenous phospholipase A₂ inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A₂ (PLA₂) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA₂. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.     

A novel hepatopancreatic phospholipase A2 from Hexaplex trunculus with digestive and toxic activities

A marine snail digestive phospholipase A₂ (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A₂, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 °C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6 mM CaCl₂. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases.Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA₂. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A₂ from venoms and another class of non toxic pancreatic phospholipase A₂ from mammals.     

Characterisation of a plasma membrane-associated phospholipase A2 activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A₂ (EC 3.1.1.4; PLA₂) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA₂ activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca²⁺, with full enzyme activity at 10 μmol/L Ca²⁺. The Ca²⁺-dependent PLA₂ activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca²⁺-dependent PLA₂ activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA₂ activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA₂ in cold acclimation in plants.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.