An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2 (PLA2) in tobacco hornworms Manduca sexta

The entomopathogenic bacterium, Xenorhabdus nematophila, induces immunodepression in target insects and finally leads to lethal septicemia of the infected hosts. A hypothesis has been raised that the bacteria inhibit eicosanoid-biosynthesis pathway to interrupt immune signaling of the infected hosts. Here, we show direct evidence that X. nematophila inhibits the activity of phospholipase A2 (PLA2), the initial step in the eicosanoid-biosynthesis pathway. Inhibition of PLA2 was dependent on both incubation time with X. nematophila and the bacterial concentration in in vitro PLA2 preparations of Manduca sexta hemocytes. While living bacteria inhibited PLA2 activity, heat-killed X. nematophila rather increased PLA2 activity. X. nematophila secreted PLA2 inhibitor(s) which were detected in the organic, but not aqueous, extract of the bacterial culture medium. The PLA2 inhibitory activity of the organic extract was lost after heat treatment. These results clearly indicate that X. nematophila inhibits PLA2 activity, and thereby inhibits eicosanoid biosynthesis which leads to immunodepression of the infected hosts.     

Three metabolites from an entomopathogenic bacterium,Xenorhabdus nematophila,inhibit larval development of Spodoptera exigua (Lepidoptera: Noctuidae) by inhibiting a digestive enzyme,phospholipase A2

Abstract An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune‐associated phospholipase A₂ (PLA₂), which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline‐tyrosine and acetylated phenylalanine‐glycine‐valine, which are known to inhibit enzyme activity of PLA₂ extracted from hemocyte and fat body. This study tested their effects on digestive PLA₂ of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 μg/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA₂ extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA₂ of S. exigua midgut may explain some of their oral toxic effects.     

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities

The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A₂ (PLA₂) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1 μg/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10 μg/insect) or PAF (1 μg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA₂ activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.     

Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua

Xenorhabdus nematophilus is a Gram-negative symbiotic bacterium of the entomopathogenic nematode, Steinernema carpocapsae. The bacteria delivered into the insect hemocoel by the nematodes cause immunodepression of the target insects to protect host nematodes and themselves from the cellular immune reaction. Previous reports suggest that the immunodepression is caused by inhibition of the eicosanoid pathway that is known to be critically important to mediate cellular immunity. This study focused on the inhibitory effect of X. nematophilus on PLA2 activity of Spodoptera exigua. The PLA2 activity was functionally associated with the activation cascade of prophenoloxidase (pPO). Dexamethasone (DEX), a specific PLA2 inhibitor, inhibited pPO activation completely at the higher doses of approximately 2.4 muM in vitro condition. The inhibitory effect of DEX was reversed by the addition of arachidonic acid, the catalytic product of PLA2. By means of this in vitro PLA2 inhibitor assay system, two different PLA2 inhibitors were used to compare their inhibitory effects on the hemolymph PLA2 of S. exigua. p-Bromophenacyl bromide (BPB), a specific inhibitor of secretory PLA2 (sPLA2), significantly inhibited pPO activation, but methylarachidonyl fluorophosphates (MAFP), a specific inhibitor of cytosolic PLA2 (cPLA2), did not show any inhibitory effect. BPB also inhibited pPO activation of the plasma, though much higher PO activation and its inhibition by BPB was found in the hemocytes. Growth medium of X. nematophilus at the stationary phase had a PLA2 inhibitory effect. Via the in vitro PLA2 inhibitor assay, it was shown that the ethyl ether extract of the medium contained significant PLA2 inhibitor activity. These results indicate that X. nematophilus produces and secretes PLA2 inhibitor, which acts on BPB-susceptible PLA2 of S. exigua.     

Surface antigens of Xenorhabdus nematophila (F. Enterobacteriaceae) and Bacillus subtilis (F. Bacillaceae) react with antibacterial factors of Malacosoma disstria (C. Insecta: O. Lepidoptera) hemolymph

Previous research established different interactions of the insect pathogen, Xenorhabdus nematophila and nonpathogen, Bacillus subtilis, with antimicrobial hemocytes and humoral factors of larval Malacosoma disstria [Giannoulis, P., Brooks, C.L., Dunphy, G.B., Mandato, C.A., Niven, D.F., Zakarian, R.J., 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobacteriaceae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasicocampidae). J. Invertebr. Pathol. 94, 20-30]. The antimicrobial systems were inhibited by X. nematophila and stimulated by B. subtilis. The bacterial surface antigens participating in these reactions were unknown. Thus, herein the effects of lipopolysaccharide (endotoxin) from X. nematophila and lipoteichoic acid from B. subtilis on the larval M. disstria immune factors, the hemocytes and phenoloxidase, were determined. Endotoxin elevated the level of damaged hemocytes limiting the removal of X. nematophila from the hemolymph and enhancing the rapid release of bacteria trapped by nodulation. Similar effects were observed with the lipid A moiety of the endotoxin. The effects of lipopolysaccharide and lipid A on the hemocyte activities were abrogated by polymyxin B (an antibiotic that binds to lipid A) confirming lipopolysaccharide as the hemocytotoxin by virtue of the lipid A moiety. Lipoteichoic acid elicited nodulation and enhanced phenoloxidase activation and/or activity. Although lipoidal endotoxin and lipid A inhibited phenoloxidase activation they enhanced the activity of the enzyme. Apolipophorin-III precluded the effects of lipopolysaccharide, lipid A, and lipoteichoic acid on the hemocytes and prophenoloxidase until the antigens exceeded a critical threshold.     

Identification of beta-type phospholipase A(2) inhibitor in a nonvenomous snake,Elaphe quadrivirgata

A novel serum protein inhibiting specifically the enzymatic activity of the basic phospholipase A(2) (PLA(2)) from the venom of the Chinese mamushi snake (Agkistrodon blomhoffii siniticus) was purified from a nonvenomous Colubridae snake, Elaphe quadrivirgata. The purified inhibitor was a 150-kDa glycoprotein having a trimeric structure, composed of two homologous 50-kDa subunits. Their amino acid sequences, containing leucine-rich repeats, were typical of the beta-type PLA(2) inhibitor (PLIbeta), previously identified from the serum of A. blomhoffii siniticus. The inhibitor inhibited exclusively group II basic PLA(2)s and did not inhibit other kinds of PLA(2)s. This is the first paper reporting the existence of PLIbeta in a nonvenomous snake. The existence of PLIbeta in the nonvenomous snake reflects that PLIbetas are widely distributed over the snake species and participate commonly in regulating the physiological activities of the unidentified target PLA(2)s.     

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.     

Asp49 phospholipase A(2)-elaidoylamide complex: a new mode of inhibition

The inhibition of phospholipase A(2)s (PLA(2)s) is of pharmacological and therapeutic interest because these enzymes are involved in several inflammatory diseases. Elaidoylamide is a powerful inhibitor of a neurotoxic PLA(2) from the Vipera ammodytes meridionalis venom. The X-ray structure of the enzyme-inhibitor complex reveals a new mode of Asp49 PLA(2) inhibition by a fatty acid hydrocarbon chain. The structure contains two identical homodimers in the asymmetric unit. In each dimer one subunit is rotated by 180 degrees with respect to the other and the two molecules are oriented head-to-tail. One molecule of elaidoylamide is bound simultaneously to the substrate binding sites of two associated neurotoxic phospholipase A(2) molecules. The inhibitor binds symmetrically to the hydrophobic channels of the two monomers. The structure can be used to design anti-inflammatory drugs.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

PGE(2) induces oenocytoid cell lysis via a G protein-coupled receptor in the beet armyworm, Spodoptera exigua

Eicosanoids mediate cellular and humoral immune responses in the beet armyworm, Spodoptera exigua, including activation of prophenoloxidase (PPO). PPO activation begins with release of its inactive zymogen, PPO, from oenocytoids in response to prostaglandins (PGs). Based on the biomedical literature, we hypothesized that PGs exert their actions via specific G protein-coupled receptor(s) in S. exigua. This study reports a G protein-coupled receptor (Se-hcPGGPCR1) gene, which is expressed in the hemocytes of S. exigua. The Se-hcPGGPCR1 consists of 420 amino acids and belongs to rhodopsin-type GPCRs. The high content of hydrophobic amino acid residues within the Se-hcPGGPCR1 protein is explained by prediction of seven-transmembrane domains that are characteristic of these GPCRs. Except for the eggs, Se-hcPGGPCR1 was expressed in all life stages. During the larval stage, it was expressed in hemocytes and gut, but not in fat body nor in epidermis. Real time quantitative RT-PCR showed that bacterial challenge induced more than 20-fold increases in its expression level. Fluorescence in situ hybridization showed that Se-hcPGGPCR1 was expressed in a specific hemocyte type, the oenocytoids. A specific eicosanoid, PGE₂, significantly induced oenocytoid lysis and increased PO activity in the plasma. In contrast, when Se-hcPGGPCR1 expression was suppressed by RNA interference (RNAi), the oenocytoid lysis and the PO activation in response to PGE₂ were not elevated above basal levels. A binding assay using intracellular calcium mobilization showed that the RNAi-treated hemocytes were significantly less responsive to PGE₂ than the control hemocytes. These results support our hypothesis with the specific finding that PGE₂ acts through Se-hcPGGPCR1 to activate PPO by lysing oenocytoids.     

Role of a small G protein Ras in cellular immune response of the beet armyworm, Spodoptera exigua

Insect cellular immune responses accompany cytoskeletal rearrangement of hemocytes to exhibit filopodial and pseudopodial extension of their cytoplasm. Small G proteins are postulated to be implicated in the hemocyte cellular processes to perform phagocytosis, nodulation, and encapsulation behaviors. A small G protein ras gene (Se-Ras) was cloned from cDNAs prepared from hemocytes of the beet armyworm, Spodoptera exigua. The open reading frame of Se-Ras encoded 179 amino acids with a predicted molecular weight of 20.0 kDa, in which 114 residues at amino terminus were predicted to be a GTP binding domain. It showed high sequence similarities (86.1-92.8%) with known ras genes in other insects. Se-Ras was constitutively expressed in all developmental stages from egg to adult without any significant change in expression levels in response to bacterial challenge. A specific double strand RNA (dsRNA) could knockdown its expression in the hemocytes after 48 h post-injection. While the RNA interference (RNAi) did not show any change in total or differential hemocyte counts, it impaired hemocyte behaviors. The RNAi of Se-Ras significantly suppressed hemocyte spreading, cytoskeleton extension, and nodulation behaviors in response to bacterial challenge. Release of prophenoloxidase from oenocytoids was significantly inhibited by the RNAi, which resulted in significant suppression in PO activation in response to an inducer, PGE₂. These results suggest that Se-Ras is implicated in mediating cellular processes of S. exigua hemocytes. This is the first report of Ras role in insect cellular immune response.     

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.     

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.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

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.