Lys-49 Phospholipases A2 Research Articles

Comparative in vitro and in silico analysis of the ability of basic Asp49 phospholipase A2 and Lys49-phospholipase A2-like myotoxins from Bothrops diporus venom to inhibit the metastatic potential of murine mammary tumor cells and endothelial cell tubulogenesis: Asp49 vs Lys49 phospholipases A2: Inhibition of metastasis and angiogenesis

Snake venoms are a complex mixture of proteins and polypeptides that represent a valuable source of potential molecular tools for understanding physiological processes for the development of new drugs. In this study two major PLA2s, named PLA2-I (Asp49) and PLA2-II (Lys49), isolated from the venom of Bothrops diporus from Northeastern Argentina, have shown cytotoxic effects on LM3 murine mammary tumor cells, with PLA2-II-like exhibiting a stronger effect compared to PLA2-I. At sub-cytotoxic levels, both PLA2s inhibited adhesion, migration, and invasion of these adenocarcinoma cells. Moreover, these toxins hindered tubulogenesis in endothelial cells, implicating a potential role in inhibiting tumor angiogenesis. All these inhibitory effects were more pronounced for the catalytically-inactive toxin. Additionally, in silico studies strongly suggest that this PLA2-II-like myotoxin could effectively block fibronectin binding to the integrin receptor, offering a dual advantage over PLA2-I in interacting with the αVβ3 integrin. In conclusion, this study reports for the first time, integrating both in vitro and in silico approaches, a comparative analysis of the antimetastatic and antiangiogenic potential effects of two isoforms, an Asp49 PLA2-I and a Lys49 PLA2-II-like, both isolated from Bothrops diporus venom.

PLA2-MjTX-II from Bothrops moojeni snake venom exhibits antimetastatic and antiangiogenic effects on human lung cancer cells

Phospholipases A2 (PLA2s) from snake venom possess antitumor and antiangiogenic properties. In this study, we evaluated the antimetastatic and antiangiogenic effects of MjTX-II, a Lys49 PLA2 isolated from Bothrops moojeni venom, on lung cancer and endothelial cells. Using in vitro and ex vivo approaches, we demonstrated that MjTX-II reduced cell proliferation and inhibited fundamental processes for lung cancer cells (A549) growth and metastasis, such as adhesion, migration, invasion, and actin cytoskeleton decrease, without significantly interfering with non-tumorigenic lung cells (BEAS-2B). Furthermore, MjTX-II caused cell cycle alterations, increased reactive oxygen species production, modulated the expression of pro- and antiangiogenic genes, and decreased vascular endothelial growth factor (VEGF) expression in HUVECs. Finally, MjTX-II inhibited ex vivo angiogenesis processes in an aortic ring model. Therefore, we conclude that MjTX-II exhibits antimetastatic and antiangiogenic effects in vitro and ex vivo and represents a molecule that hold promise as a pharmacological model for antitumor therapy.

Antiangiogenic effects of phospholipase A2 Lys49 BnSP-7 from Bothrops pauloensis snake venom on endothelial cells: An in vitro and ex vivo approach

Antiangiogenic strategies are promising tools for cancer treatment and several other disorders. In this sense, phospholipases A2 (PLA2s) from snake venom have been described to possess antiangiogenic properties. In this study, we evaluated both in vitro and ex vivo antiangiogenic effects induced by BnSP-7, a Lys49 PLA2 isolated from Bothrops pauloensis snake venom. BnSP-7 was able to inhibit endothelial cell (HUVEC) proliferation, which was indeed confirmed by a modulation of cell cycle progression. Interestingly, BnSP-7 also inhibited the adhesion and migration of HUVECs and blocked in vitro angiogenesis in a VEGF-dependent manner, an important proangiogenic factor. Finally, BnSP-7 was capable of inhibiting sprouting angiogenic process through an ex vivo aortic ring assay. Taken together, these results indicate that BnSP-7 has potent in vitro and ex vivo antiangiogenic effect.

Bothrops pauloensis snake venom-derived Asp-49 and Lys-49 phospholipases A2 mediates acute kidney injury by oxidative stress and release of inflammatory cytokines

The envenomation caused by the Bothrops pauloensis snake leads to severe local and systemic effects including acute kidney injury. In this study, we investigated the renal effects by phospholipases A2 (PLA2s), divided into two main subgroups, Asp-49 and Lys-49, isolated from the Bothrops pauloensis snake venom (BpV) in isolated rat kidney system. Both PLA2s (3 μg/mL), added alone to the perfusion system and analyzed for 120 min, had significant effects on isolated rat kidney. Asp-49 reduced Glomerular Filtration Rate (GFR) at 60, 90 and 120 min, and the percentage of total tubular sodium transport (%TNa+) and potassium transport (%TK+) at 120 min. Lys-49 increased Perfusion Pressure (PP) at 120 min and reduced GFR, %TNa+ and the percentage of total tubular chloride transport (%TCl−) at 60, 90 and 120 min. Cytokine release in the kidney tissues were increased with Asp-49 PLA2 (IL-10) and Lys-49 PLA2 (TNF-α, IL-1β, IL-10). Both increased MPO activity. Asp-49 PLA2 decreased Glutathione (GSH) and increased nitrite levels, while Lys-49 PLA2 increased Malondialdehyde (MDA), GSH and nitrite levels. Histological analysis of the perfused kidneys revealed the presence of glomerular degeneration and atrophy, deposit of proteinaceous material in Bowman's space and intratubular with both PLA2s. These findings indicated that both PLA2s modified the functional parameters in an isolated perfused kidney model with increased oxidative stress and cytokine release. PLA2s are one of the components at high concentration in BpV and our results provide important knowledge about their involvement with the nephrotoxic mechanism.

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2plasma inhibitor from the non-venomous Brazilian snakeBoa constrictor.

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

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening.

Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

Conservation analysis and decomposition of residue correlation networks in the phospholipase A2 superfamily (PLA2s): Insights into the structure-function relationships of snake venom toxins

Phospholipases A2 (PLA2s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA2s family with special emphasis on snake venom PLA2s (svPLA2s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA2s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA2s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for important positions in the family of PLA2s, and potential new objects of investigation.

BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential

Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100μg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.

Photobiostimulation reduces edema formation induced in mice by Lys-49 phospholipases A2 isolated from Bothrops moojeni venom.

The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.

Inhibitory effects of Swietenia macrophylla on myotoxic phospholipases A2

Activity-guided fractionation of an ethanol-soluble extract of the leaves of Swietenia macrophylla King, Meliaceae, led to several fractions. As a result, sample Sm13-16, 23 had the most promising activity against phospholipases A2 (PLA2), Asp49 and Lys49 types. This fraction inhibited PLA2 activity of the Asp49 PLA2, when aggregated substrate was used. On the other hand, this activity was weakly neutralized when monodispersed substrate was used. In addition, Sm13-16, 23 inhibited, in a dose dependent manner, the cytotoxicity, myotoxicity and edema induced by PLA2s, as well as the anticoagulant activity of Asp49 PLA2. Overall, this fraction exhibited a better inhibition of the toxic activities induced by the Lys49 PLA2 than those caused by the Asp49 PLA2. The spectral data of Sm13-16, 23 suggested the presence of aromatic compounds (UV λ max (nm) 655, 266, and 219; IR λ max KBr (cm−1): ~ 3600-3000 (OH), 2923.07 and 1438.90 (C-H), 1656.69 (C = O), 1618.63 and 1607.67 (C-O), 1285.47-772.60). We suggest that phenolic compounds could interact and inhibit the toxins by several mechanisms. Further analysis of the compounds present in the active fraction could be a relevant contribution in the treatment of accidents caused by snake envenomation.

Molecular Characterization of Lys49 and Asp49 Phospholipases A2 from Snake Venom and Their Antiviral Activities against Dengue virus

We report the detailed molecular characterization of two PLA2s, Lys49 and Asp49 isolated from Bothrops leucurus venom, and examined their effects against Dengue virus (DENV). The Bl-PLA2s, named BlK-PLA2 and BlD-PLA2, are composed of 121 and 122 amino acids determined by automated sequencing of the native proteins and peptides produced by digestion with trypsin. They contain fourteen cysteines with pIs of 9.05 and 8.18 for BlK- and BlD-PLA2s, and show a high degree of sequence similarity to homologous snake venom PLA2s, but may display different biological effects. Molecular masses of 13,689.220 (Lys49) and 13,978.386 (Asp49) were determined by mass spectrometry. DENV causes a prevalent arboviral disease in humans, and no clinically approved antiviral therapy is currently available to treat DENV infections. The maximum non-toxic concentration of the proteins to LLC-MK2 cells determined by MTT assay was 40 µg/mL for Bl-PLA2s (pool) and 20 µg/mL for each isoform. Antiviral effects of Bl-PLA2s were assessed by quantitative Real-Time PCR. Bl-PLA2s were able to reduce DENV-1, DENV-2, and DENV-3 serotypes in LLC-MK2 cells infection. Our data provide further insight into the structural properties and their antiviral activity against DENV, opening up possibilities for biotechnological applications of these Bl-PLA2s as tools of research.

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional–structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s.

Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin – a Novel Feature in Lys49-PLA2s Protein Class

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys.

Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom.

Two myotoxic and noncatalytic Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A(2) (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56-2.05 Å and belonged to space groups P3(1)21 (braziliantoxin-II), P6(5)22 (braziliantoxin-III) and P2(1) (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A(2) braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A(2).

Comparative structural studies on Lys49-phospholipases A2 from Bothrops genus reveal their myotoxic site

Phospholipases A(2) (PLA(2)s) are membrane-associated enzymes that hydrolyze phospholipids at the sn-2 position, releasing lysophospholipids and free fatty acids. Phospholipase A(2) homologues (Lys49-PLA(2)s) are highly myotoxic and cause extensive tissue damage despite not showing measurable catalytic activity. They are found in different snake venoms and represent one third of bothropic venom composition. The importance of these toxins during envenomation is related to the pronounced local myotoxic effect they induce since this effect is not neutralized by serum therapy. We present herein three structures of Lys49-PLA(2)s from Bothrops genus snake venom crystallized under the same conditions, two of which were grown in the presence of alpha-tocopherol (vitamin E). Comparative structural analysis of these and other Lys49-PLA(2)s showed two different patterns of oligomeric conformation that are related to the presence or absence of ligands in the hydrophobic channel. This work also confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. For the first time, a residue from the short-helix (Lys20) is suggested as a member of this site and the importance of Tyr119 residue to myotoxicity of bothropic Lys49-PLA(2)s is also discussed. These results support a complete hypothesis for these PLA(2)s myotoxic activity consistent with all findings on bothropic Lys49-PLA(2)s studied up to this moment, including crystallographic, bioinformatics, biochemical and biophysical data.

Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries.

Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species.

Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A2: quaternary structure and inhibition mechanism insights

Phospholipases A2 are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. A class of PLA2-like proteins has been described which despite PLA2 activity on artificial substrate, due to a D49K mutation, is still highly myonecrotic. This work reports the X-ray structure determination of two Lys49-PLA2s from Bothrops neuwiedi pauloensis (BnSP-7 and BnSP-6) and, for the first time, the comparison of eight dimeric Lys49-PLA2s. This comparison reveals that there are not just two (“open” and “closed”) but at least six different conformations. The binding of fatty acid observed in three recent Lys49-PLA2 structures seems to be independent of their quaternary conformation. Cys29 polarization by Lys122 is not significant for BnSP-7 and BnSP-6 or other structures not bound by fatty acids. These structures may be in an “active” state when nothing is bound to them and the Lys122/Cys29 interactions are weak or absent.

Purification, Sequencing, and Phylogenetic Analyses of Novel Lys-49 Phospholipases A2 from the Venoms of Rattlesnakes and other Pit Vipers

Basic phospholipase A2 homologs with Lys49 substitution at the essential Ca2+-binding site are present in the venom of pit vipers under many genera. However, they have not been found in rattlesnake venoms before. We have now screened for this protein in the venom of rattlesnakes and other less studied pit vipers. By gel filtration chromatography and RP-HPLC, Lys49-phospholipase-like proteins were purified from the venoms of two rattlers, Crotalus atrox and Crotalus m. molossus, and five nonrattlers, Porthidium nummifer, Porthidium godmani, Bothriechis schlegelii, Trimeresurus puniceus, and Trimeresurus albolabris. Their N-terminal amino acid sequences were shown to be characteristic for this phospholipase subfamily. The purified basic proteins from rattlesnakes caused myonecrosis and edema in experimental animals. We have also cloned the cDNAs and solved the complete sequences of four novel Lys49-phospholipases from the venom glands of C. atrox, P. godmani, B. schlegelii, and Deinagkistrodon acutus (hundred-pace). Phylogenetic analyses based on the amino acid sequences of 28 Lys49-phospholipases separate the pitviper of the New World from those of the Old World, and the arboreal Asiatic species from the terrestrial Asiatic species. The implications of the phylogeny tree to the systematics of pit vipers, and structure–function relationship of the Lys49-phospholipases are discussed.

Modulation of the Susceptibility of Human Erythrocytes to Snake Venom Myotoxic Phospholipases A2: Role of Negatively Charged Phospholipids as Potential Membrane Binding Sites

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp-49 and Lys-49 phospholipases A2 (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.

Dissociation of Enzymatic and Pharmacological Properties of Piratoxins-I and -III, Two Myotoxic Phospholipases A2 from Bothrops pirajai Snake Venom

Piratoxins (PrTX) I and III are phospholipases A2 (PLA2s) or PLA2 homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA2, while PrTX-I is a Lys49 PLA2 homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities.