Venom PLA Research Articles

Electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase A2

Results from high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) coupled to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) indicated that the monomer and dimer of phospholipase A(2) (PLA(2)) coexisted in crude Chinese Agkistrodon blomhoffii Ussurensis snake venom (ABUSV). Then, an acidic PLA(2) with the accurate molecular mass of 13979.6 Da was purified from ABUSV (mo-ABUSV-aPLA(2)). MS/MS-derived peptides from ABUSV-aPLA(2) were compared with other homologous snake venom PLA(2)s, which in turn showed that ABUSV-aPLA(2) is a novel snake venom PLA(2). Meanwhile, the ABUSV-aPLA(2) dimer (di-ABUSV-aPLA(2)) was also obtained. MS/MS analysis identified the same peptides from di-ABUSV-aPLA(2) as from mo-ABUSV-aPLA(2), which indicates that di-ABUSV-aPLA(2) is a homodimer. One Ca(2+) ion is contained per ABUSV-aPLA(2). The Ca(2+) ion is critical for both the hydrolytic activity and the structure of ABUSV-aPLA(2). Pro-Q Emerald and Pro-Q Diamond specific glycoprotein and phosphoprotein staining combined with MS/MS analysis indicated that the ABUSV-aPLA(2) is both a glycoprotein and a phosphoprotein, which to our knowledge is the first such report for a snake venom PLA(2) and thus provides new threads for the study of the functions and structures of snake venom PLA(2)s. One phosphorylation site and the size of the glycan chain are determined by using HPLC/nESI-MS/MS and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. The delicate utilization of ESI-MS can exert tremendous impact on protein sciences.

Activation of phospholipase A 2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero- sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero- sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.

Fluorescence Single‐Molecule Study of Cobra Phospholipase A2 Action on a Supported Gel‐Phase Lipid Bilayer

Previous nanoscale investigations of the gel-state membrane surface structure under the action of phospholipase A(2) (PLA(2)) suggest that single enzymes at work scoot on the membrane surface from the observed defects, which creates nanosized channels oriented along the lipid crystal-packing structure. To date, however, there have been no reports of direct observation of PLA(2) at the single-molecule level focusing on how the enzymes interact with the defects. Herein, we report a single-molecule fluorescence microscopy study on the action of enzymatically active rhodamine B-labeled cobra PLA(2) on a supported lipid membrane with visible packing defects on a glass substrate. Working with a gel-state phospholipid bilayer, the low-activity period (lag phase) of PLA(2) action is followed by the burst binding of PLA(2) molecules from aqueous solution on a few newly created active sites. These active sites are distinguished by a spatial resolution of approximately 40 nm, which is below the diffraction limit. The rate of active-site propagation as reflected by new PLA(2) binding on the membrane surface is estimated to be approximately 5 nm min(-1). This rate is about two orders of magnitude slower than the propagation rate of hydrolyzed channels estimated by AFM studies on bee venom PLA(2) on a similar membrane surface. This direct observation of PLA(2) molecules allows the visualization of different PLA(2) binding modes on the membrane surface and on the membrane boundary.

Analyses of Group III Secreted Phospholipase A2 Transgenic Mice Reveal Potential Participation of This Enzyme in Plasma Lipoprotein Modification, Macrophage Foam Cell Formation, and Atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.

Effects of petrosaspongiolide R on the surface topology of bee venom PLA 2: A limited proteolysis and mass spectrometry analysis

Petrosaspongiolides are sponge metabolites belonging to the family of the γ-hydroxybutenolide marine terpenoids. They possess a remarkable in vitro and in vivo anti-inflammatory profile, due to the specific inhibition of group II and III secretory phospholipase A 2 enzymes, and for this reason can be considered as potential lead for the development of anti-inflammatory drugs. The molecular mechanism of bee venom phospholipase A 2 inactivation has been identified, and the ligand–enzyme complex formation is guided by either non-covalent and covalent interactions. In this work we have analyzed the conformational changes induced by petrosaspongiolide R on the bee venom phospholipase A 2 topology during the molecular recognition process, through the application of limited proteolysis and mass spectrometric methodologies. The results are indicative of structural changes at the N- and C-terminal domains producing a more compact conformational arrangement of the enzyme.

Mitochondrial dysfunction induced by pancreatic and crotalic ( Crotalus durissus terrificus) phospholipases A 2 on rabbit proximal tubules suspensions

In the present study we show that phospholipases A 2 isolated from porcine pancreas (PP-PLA 2) and Crotalus durissus terrificus snake venom (SV-PLA 2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA 2s induced increases in non-esterified fatty acid (NEFA) levels (μg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA 2, and were dose-dependent for the crotalic PLA 2 group. Furthermore, snake venom PLA 2 induced a decrease in mitochondrial membrane potential (ΔΨ m) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA 2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on ΔΨ m measured with safranin O. Incubation with pancreatic and crotalic phospholipases A 2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.

Secretory phospholipases A 2 isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E 2 and D 2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A 2 (sPLA 2) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA 2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA 2s promoted the synthesis of PGD 2 and PGE 2, with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA 2s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. Inhibition of cPLA 2 by AACOCF 3, but not of iPLA 2 by PACOCF 3 or BEL, significantly reduced PGD 2, PGE 2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA 2 inhibition did not modify the effects of CB, whereas iPLA 2 inhibition reduced PGD 2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs' synthesis are triggered by these snake venom sPLA 2s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA 2s in vivo.

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action.

A large variety of snake toxins evolved from PLA(2) digestive enzymes through a process of 'accelerated evolution'. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA(2)s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca(2+) concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells.

Mapping the structural determinants of presynaptic neurotoxicity of snake venom phospholipases A 2

The structural features of presynaptically neurotoxic secretory phospholipases A 2 (sPLA 2s) that are responsible for their potent and specific action are still a matter of debate. To identify the residues that distinguish a highly neurotoxic sPLA 2, ammodytoxin A (AtxA), from a structurally similar but more than two orders of magnitude less toxic Russell's viper sPLA 2, VIIIa, we prepared a range of mutants and compared their properties. The results show that the structural features that confer high neurotoxicity to AtxA extend from its C-terminal part, with a central role of the residues Y115, I116, R118, N119 (the YIRN cluster) and F124, across the interfacial binding surface (IBS) in the vicinity of F24, to the N-terminal helix whose residues M7 and G11 are located on the edges of the IBS. Competition binding studies indicate that the surface of interaction with the neuronal M-type sPLA 2 receptor R180 extends over a similar region of the molecule. In addition, the YIRN cluster of AtxA is crucial for the high-affinity interaction with two intracellular binding proteins, calmodulin and R25. The concept of a single “presynaptic neurotoxic site” on the surface of snake venom sPLA 2s is not consistent with these results which suggest that different parts of the toxin molecule are involved in distinct steps of presynaptic neurotoxicity.

Secretory PLA2 inhibitor indoxam suppresses LDL modification and associated inflammatory responses in TNFα‐stimulated human endothelial cells

Secretory phospholipase A2 (sPLA2) is implicated in atherosclerosis, although the effects of specific sPLA2 inhibitors have not been studied. We investigated the effects of the indole analogue indoxam on low-density lipoprotein (LDL) modification by sPLA2 enzymes of different types and on the associated inflammatory responses in human umbilical vein endothelial cells (HUVEC). LDL modification was assessed by measuring the contents of two major molecular species of lysophosphatidylcholine (LPC) using electrospray ionization-liquid chromatography/mass spectrometry. The proinflammatory activity of the modified LDL was evaluated by determining monocyte chemoattractant protein-1 (MCP-1) mRNA expression and transcriptional factor nuclear factor-kappaB (NF-kappaB) activity in HUVEC. Indoxam dose-dependently inhibited palmitoyl- and stearoyl-LPC production in LDL incubated with snake venom sPLA2 (IC50 1.2 microM for palmitoyl-LPC, 0.8 microM for stearoyl-LPC). MCP-1 mRNA expression and NF-kappaB activity were enhanced by venom sPLA2-treated LDL, which was completely suppressed by indoxam but not by thioetheramide-PC, a competitive sPLA2 inhibitor. Indoxam also suppressed LPC production in LDL treated with human synovial type IIA sPLA2. Tumour necrosis factor alpha (TNFalpha) increased type V sPLA2 expression in HUVEC. Indoxam dose-dependently suppressed LPC production in native and glycoxidized LDL treated with TNFalpha-stimulated HUVEC. Indoxam suppressed MCP-1 mRNA expression and NF-kappaB activity in TNFalpha-stimulated HUVEC incubated with native or glycoxidized LDL. Indoxam prevented sPLA2-induced LPC production in native and glycoxidized LDL as well as LDL-induced inflammatory activity in HUVEC. Our results suggest that indoxam may be a potentially useful anti-atherogenic agent.

Isolation of a snake venom phospholipase A 2 (PLA 2) inhibitor (AIPLAI) from leaves of Azadirachta indica ( Neem): Mechanism of PLA 2 inhibition by AIPLAI in vitro condition

A compound (AIPLAI ( Azadirachta indica PLA 2 inhibitor)) purified from the methanolic leaf extract of A. indica ( Neem) inhibits the cobra and Russell's viper venoms (RVVs) phospholipase A 2 enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom ( Naja naja and Naja kaouthia) PLA 2 enzymes by AIPLAI is significantly higher ( P<0.05) compared to the inhibition of PLA 2 enzymes of crude RVV ( Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA 2 enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.

The effects of bee ( Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml −1 bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10 −12 mg ml −1) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10 −5–10 −6 mg ml −1. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.

Crystal structure of Natratoxin, a novel snake secreted phospholipaseA2 neurotoxin from Naja atra venom inhibiting A‐type K+ currents

Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold.

Gangliosides as potential inhibitors of Naja naja venom PLA 2 (NV-PLA 2 ) induced human erythrocyte membrane damage

We determined the ability of mixed gangliosides (16% GD1b, 19% GT1b, 21% GM1, and 40% GD1a) and individual gangliosides GM1 and GD1b to modulate the NV-PLA2 induced human erythrocyte ghost membrane damage. CM-Sephadex purification of crude Naja naja venom yielded eight peaks of which peak VII, a major phospholipase A2 (NV-PLA2) accounted for 22% of the total protein recovered and 8% of the total PLA2 activity recovered. The membrane damage induced by NV-PLA2 was assessed by measuring the decrease in the relative intensity of fluorescence using cis–parinaric acid (PnA) as a monitor molecule. The RBC membranes isolated from healthy human blood showed 72% damage on treatment with NV-PLA2 (2 mg) when compared to untreated membranes. Mixed gangliosides (18 nM) and GM1 (15 nM) offered 81 and 86% protection respectively, whereas GD1b (20 nM) did not show significant protection. Analysis of membrane bound Na+K+ and Ca2+Mg2+ ATPase indicated a 3 fold and 2 folds decrease in their activities on NV-PLA2 treatment when compared to untreated membranes. Mixed gangliosides restored the Na+K+ ATPase activity by 78%, whereas GM1 and GD1b offered 74 and 52% restoration respectively. The Ca2+Mg2+ ATPase activity was restored by 80 and 81% with mixed gangliosides and GM1 respectively. GD1b showed only 50% restoration. Mixed gangliosides and GM1 exhibited a significant dose dependent inhibition of NV-PLA2 activity when compared to GD1b. The data show that mixed gangliosides and GM1 were effective in modulating NV-PLA2induced erythrocyte membrane damage than GD1b. Key words: Gangliosides, NV-PLA2, PnA, gangliosides, erythrocyte membrane, Na+ K+ ATPase, Ca2+ Mg2+ ATPase.

Reappraisal of Vipera aspis Venom Neurotoxicity

BackgroundThe variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA2 neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA2 composition of the snakes captured in the same areas.Methodology/Principal FindingsWe conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA2s. We used SELDI technology to study the diversity of PLA2 in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA2s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA2 venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains.Conclusions/SignificanceThe association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA2 genome and transcriptome data.

The oral absorption of phospholipid prodrugs: In vivo and in vitro mechanistic investigation of trafficking of a lecithin-valproic acid conjugate following oral administration

The purpose of this study was to evaluate the oral absorption characteristics of a phospholipid–drug conjugate, comprising direct conjugation between the lecithin and the drug moiety through the sn-2 position. We investigated the mechanisms involved with the trafficking of this conjugate following oral administration in the gastrointestinal (GI) lumen, within the enterocyte and further. A phospholipid-valproic acid conjugate (DP-VPA) was utilized as a model molecule. The oral absorption of this conjugate in rats was investigated following administration in long (LCT) vs. medium (MCT) chain triglyceride formulations, and in the postprandial vs. fasted state. Oral administration within the LCT solution caused more than a 3-fold increase in DP-VPA bioavailability in comparison to the MCT solution. Moreover, a significant food effect was evident for DP-VPA. Hence, we evaluated the lymphatic transport of DP-VPA in mesenteric lymph duct cannulated freely moving rats. Sixty percent of the absorbed DP-VPA was associated with lymphatic transport. Similar DP-VPA absorption was obtained in secretory type II PLA 2 knockout mice (C57BL/6) and in control mice (BALB/c). Moreover, nil DP-VPA degradation in serum and very low (4.8%) degradation by bee venom PLA 2 in vitro were obtained. In conclusion, direct conjugation between the drug and the phospholipid produces a complex having unique absorption properties that include: (1) a stable complex that does not undergo degradation in the GI tract; (2) permeation through the gut wall and entering intact to the enterocyte; and (3) association with chylomicron in the enterocyte and reaching the systemic circulation via the lymphatic route. These unique properties may be of interest in drug delivery.

Human group III phospholipase A 2 suppresses adenovirus infection into host cells : Evidence that group III, V and X phospholipase A 2s act on distinct cellular phospholipid molecular species

Of 10 mammalian secreted phospholipase A 2 (sPLA 2) enzymes identified to date, group V and X sPLA 2s, which are two potent plasma membrane-acting sPLA 2s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA 2, which is structurally more similar to bee venom PLA 2 than to other mammalian sPLA 2s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA 2 hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA 2 domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA 2 depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA 2s target distinct phospholipid molecular species in cellular membranes.

Scalaradial, a Dialdehyde‐Containing Marine Metabolite That Causes an Unexpected Noncovalent PLA2 Inactivation

Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti-inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A(2) (sPLA(2)). Scalaradial, a 1,4-dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti-inflammatory profile, both in vitro and in vivo, through selective sPLA(2) inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on-going research on anti-PLA(2) natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA(2) inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA(2), a model sPLA(2) that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA(2) inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand-enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti-inflammatory compounds that target sPLA(2) enzymes.

Purification, sequencing and structural characterization of the phospholipase A 1 from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA 1 was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA 1's. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA 1 is 53–82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% α-helix, 28% β-sheet and 49% coil. The protein structure has the α/ β fold common to many lipases; the core consists of a tightly packed β-sheet constituted of six-stranded parallel and one anti-parallel β-strand, surrounded by four α-helices. P. paulista PLA 1 exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA 1 was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.

Venom phospholipases of Russell's vipers from Myanmar and eastern India—Cloning, characterization and phylogeographic analysis

Venoms of Russell's vipers (genus Daboia) are known for their deadly coagulopathic and other effects. We herein studied various isoforms of venom phospholipases A 2 (PLAs) from two Daboia species at their geographic boundary. From Myanmar Daboia siamensis venom (designated as DsM), four PLAs (designated DsM-aI, aI', aII' and bI') were purified, and the cDNAs encoding two acidic (DsM-aI and aII) and two basic PLAs (DsM-bI and S1) were also cloned from its venom-glands. DsM-S1 is identical to the major venom PLA of southern India Daboia russelii, but the protein is absent from the venom. Additionally, four PLAs (designated DrK-aI, aII, bI and bII) were cloned from cDNA obtained from venom glands of a Kolkata D. russelii, and the PLAs were purified from the pooled venom (designated as DrK). The acidic DrK-aI is the most neurotoxic and lethal among these PLAs; DsM-aI which differs from DrK-aI by only the Phe2 substitution shows greatly reduced enzymatic activity and lethality. Both acidic PLAs do not form dimeric complex with basic PLAs in the same venoms. DsM-bI' is neurotoxic and lethal but its orthologous DrK-bI (97% identical to DsM-bI') is a much weaker toxin. Given the fact that most of the orthologous PLAs of DrK and DsM share 97–100% sequence identity, Daboia vipers of Myanmar and Kolkata must be closely related. Molecular phylogenetic analyses on 30 venom PLAs of Eurasian vipers' revealed co-evolution of five subtypes of venom PLAs in both Daboia and Vipera genera. Our results shed light on the intra- and inter-species variations and structure–function relationships of viperid venom PLAs.