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Abstract: Snake venom phospholipases A2 (PLA2s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA2s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram‐positive and Gram‐negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The crystal structure of an acidic phospholipase A2 isolated from Bothrops jararacussu venom (BthA-I) chemically modified with p-bromophenacyl bromide (BPB) has been determined at 1.85 Å resolution. The catalytic, platelet-aggregation inhibition, anticoagulant and hypotensive activities of BthA-I are abolished by ligand binding. Electron-density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate-binding cleft. The BthA-I–BPB complex contains three structural regions that are modified after inhibitor binding: the Ca2+-binding loop, β-wing and C-terminal regions. Comparison of BthA-I–BPB with two other BPB-inhibited PLA2 structures suggests that in the absence of Na+ ions at the Ca2+-binding loop, this loop and other regions of the PLA2s undergo structural changes. The BthA-I–BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the `pancreatic' loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA-I–BPB, leading to a new hypothesis regarding the abolition of this activity by BPB. [ABSTRACT FROM AUTHOR]

Abstract: The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C18H36O2) has been determined at a resolution of 1.8 Å. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA2s is analogous to phospholipids and transition state analogues to catalytically active PLA2s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA2s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA2s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in group I/II PLA2s, and to the possible interactions of Lys49-PLA2s with target membranes. [Copyright &y& Elsevier]

Abstract: Bothropstoxin-II (Bthtx-II), an Asp-49 phospholipase A2 (D-PLA2) isolated from Bothrops jararacussu snake venom is able to induce platelet aggregation in a concentration-dependent manner. This effect was not due to the release of ADP from platelets since the aggregation was not suppressed by ADP scavenger systems. PMSF and PPACK were unable to inhibit Bthtx-II-induced platelet aggregation. Thus, a thrombin-like proaggregating activity of Bthtx-II can be excluded as its mechanism of action. On the other hand, indomethacin at low concentrations inhibited more markedly the ATP-release reaction than the aggregation induced by Bthtx-II, indicating that generation of cyclooxigenase products is not the most important event for the platelet aggregation reaction. It was also found that staurosporine and genistein suppressed both platelet aggregation and ATP-release reactions, but not the platelet shape-change induced by Bthtx-II. Substances that either directly activates adenylyl cyclase enzyme (forskolin and PGE1) or cell-permeant increasing agents (dibutyril-cAMP) inhibited in a concentration-dependent fashion, the platelet aggregation effects induced by the protein. It is concluded that Bthtx-II induces platelet aggregation and secretion through multiple signal transduction pathways. [Copyright &y& Elsevier]

Bothrops moojeni crude venom (MjCV) and its two major toxins, namely myotoxin I (MjTX-I) and myotoxin II (MjTX-II) were alkylated by p-bromophenacyl bromide (BPB). After alkylation the i.p. LD50 (mice) of MjCV and MjTX-I/II increased from 6.0 to 15.7 mg/kg and from 8.0 to 45.0 mg/kg, respectively. In addition, doses of 5× LD50 of alkylated MjTX-I did not cause a single death in mice and no myonecrosis was detected for the alkylated toxins, although both proteins still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A2 found in snake venoms of the genus Bothrops. Myotoxic PLA2s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA2s, despite differences in their amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were apparently more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies raised against the native or modified toxins inhibited their PLA2 and myotoxic activities. Therefore, alkylation of His48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while retaining antigenicity, which suggests a promising procedure for an enhanced antiophidian serum production for practical purposes. [Copyright &y& Elsevier]

Phospholipases A2 (PLA2s) constitute major components of snake venoms and have been extensively investigated not only because they are very abundant in these venoms but mainly because they display a wide range of biological effects, including neurotoxic, myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anti-coagulant, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoral, anti-malarial and anti-parasitic. Due to this functional diversity, these structurally similar proteins aroused the interest of many researchers as molecular models for study of structure–function relationships. One of the main experimental strategies used for the study of myotoxic PLA2s is the traditional chemical modification of specific amino acid residues (His, Met, Lys, Tyr, Trp and others) and examination of the consequent effects upon the enzymatic, toxic and pharmacological activities. This line of research has provided useful insights into the structural determinants of the action of these enzymes and, together with additional strategies, supports the concept of the presence of ‘pharmacological sites’ distinct from the catalytic site in snake venom myotoxic PLA2s. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

An acidic (pI∼4.5) phospholipase A2 (BthA-I-PLA2) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an ∼13.7 kDa single chain Asp49 PLA2 with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 1SLWQFGKMINYVM–GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC51. Crystals of this acidic protein diffracted beyond 2.0 A˚ resolution. These crystals are monoclinic and have unit cell dimensions of a=33.9, b=63.8, c=49.1 A˚, and β=104.0°. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3–4 times more active than BthTX-II, a basic D49 myotoxic PLA2 from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA2 caused a hypotensive response in the rat and inhibited platelet aggregation. Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA2, while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA2 homologues and that either of these two activities may exist alone. BthA-I-PLA2, in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. [Copyright &y& Elsevier]

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A2 (PLA2) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn2+ was the most effective in reducing mouse paw edema induced by BthTX-I (25 μg). Preincubating BthTX-I with Mn2+ (1.0 mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 μg toxin. Mn2+ (50 μl of a 1 mM solution) administered within 1 min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn2+ (1.0 mM) completely prevented BthTX-I (1.4 μM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn2+ (0.25 mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 μM) for 25 h. Preincubation with 0.25 mM Mn2+ increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn2+. BthTX-I (1 μM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25 h. PLA2 activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25 mM Mn2+. Mn2+ ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA2 homologues. [Copyright &y& Elsevier]

Phospholipases A2 (PLA2s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA2s. BmooPLA2-I is an acidic, nontoxic and catalytic PLA2 isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA2-I has been crystallized and X-ray diffraction data have been collected to 1.6 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C2221, with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 Å. The molecular-replacement solution of BmooPLA2-I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA2 from B. jararacussu (BthA-I) and other toxic and nontoxic PLA2s may provide important insights into the functional aspects of this class of proteins. [ABSTRACT FROM AUTHOR]

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A2 from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 Å resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P212121, indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A2 structures belong to space group P3121, while the crystals of complexed structures belong to space groups P21 or P212121. [ABSTRACT FROM AUTHOR]

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA 2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (col CB-A and col CB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the col CB-B presents features of the class I isoforms while col CB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for col CB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms. • The crystal structure of CB from C. d. collilineatus venom was obtained. • Crystal structure is a heterodimer composed of two new CB isoforms. • Bioinformatic and biophysical analyses showed that the toxin is tetrameric. • col CB-A isoform cannot be classified into classes I and II based on its sequence. • We hypothesized that there are more than two classes of CB isoforms. [ABSTRACT FROM AUTHOR]

Abstract: Lys49 phospholipase A2 (PLA2) homologues are myotoxic proteins devoid of catalytic activity. Their toxic determinants map to the C-terminal region 115–129, which plays an effector role in membrane damage. The dimeric state was reported to be essential for a Lys49 PLA2 which lost its liposome-disrupting activity after dissociating into monomers at pH 5.0. This study, evaluated the effects of a pH-induced dissociation on the toxicity of four Lys49 PLA2s, using biological targets instead. Both their cytolytic and myotoxic activities were lower at pH 5.0 than at pH 7.2. However, in contrast with experiments using artificial bilayers, toxic effects upon biological targets were not abolished at pH 5.0. Importantly, C-terminal synthetic peptides of two Lys49 PLA2s also showed lower cytolytic action at pH 5.0 than at pH 7.2, indicating that factors other than the dimeric/monomeric state of the proteins may also be involved in these differences of toxicity. Results support the view that the dimeric state of Lys49 PLA2s could play an enhancing, although not essential role, in their C-terminal region-mediated mechanism of myotoxicity. [Copyright &y& Elsevier]

Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a K D of 9.146 × 10−7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA 2 , thus proposing a new mechanism of PLA 2 inhibition, and presenting more evidence of GA's potential as an antivenom compound. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the in vivo anti- Leishmania amazonensis activity of a Phospholipase A 2 (Asp49-PLA 2 ), isolated from Bothrops jararacussu venom, encapsulated in liposomes as a modified toxin release system. The activity of the liposomes was evaluated in BALB/c mice, previously infected with 1 × 10 5 of the parasite's promastigotes. The size of the paw lesion in Asp49-PLA 2 -liposomal-treated animals, after 21 days, was observed as decreasing by 16% relative to the untreated control group and 12% by the Glucantime®-treated animals, which was used as a reference drug. At the end of the treatment, the animals were sacrificed and the paw and lymph node tissues were collected. Part of the collection was used to recover amastigotes and another to quantify cytokines and nitrites. In the group treated with Asp49-PLA 2 -liposomes the parasitic load was observed to be reduced by 73.5% in the macerated lymph node, compared to the control group. Comparatively, in the paw tissue was observed a reduction of 57.1%. The infected groups treated with Asp49-PLA 2 -liposomes showed significant production in TNF-α measured in lymph nodes and paw (43.73 pg/mL ± 2.25 and 81.03 pg/mL ± 5.52, respectively) and nitrite levels (31.28 μM ± 0.58 and 35.64 μM ± 5.08) also measured in lymph nodes and paw tissues, respectively, compared to untreated groups. These results indicate that the Asp49-PLA 2 -loaded liposomes were able to activate the production of some cellular components of the protective T H 1 response during the infection, constituting a promising tool for inducing the microbicidal activity of the Leishmania -infected macrophages. [ABSTRACT FROM AUTHOR]

This report describes the effect of photobiomodulation (PBM) on edema formation, leukocyte influx, prostaglandin E 2 (PGE 2 ) biosynthesis and cytotoxicity caused by bothropstoxin-I (BthTX-I), a phospholipase A 2 (PLA 2 ) homologue isolated from Bothrops jararacussu snake venom. Swiss mice or C2C12 cells were irradiated with low-level laser (LLL) at 685 nm wavelength, an energy density of 4.6 J/cm 2 and an irradiation time of 13 s. To evaluate the effect on edema formation and leukocyte influx, LLL was applied to the site of inoculation 30 min and 3 h post-injection. C2C12 cells were exposed to BthTX-I and immediately irradiated. PBM significantly reduced paw edema formation, peritoneal leukocyte influx and PGE 2 synthesis, but increased the viability of C2C12 muscle cells after BthTX-I incubation. These findings demonstrate that PBM attenuated the inflammatory events induced by BthTX-I. The attenuation of PGE 2 synthesis could be an important factor in the reduced inflammatory response caused by laser irradiation. The ability of LLL irradiation to protect muscle cells against the deleterious effects of BthTX-I may indicate preservation of the plasma membrane. [ABSTRACT FROM AUTHOR]

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A 2 (PLA 2 ), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB 4 production, cytosolic PLA 2 s activity, myeloperoxidase (MPO) and superoxide anion (O 2 − ) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB 4 , but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O 2 − release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O 2 − . However, it induces LTB 4 and IL-8 production. These data show the influence of CNF on PBMCs’ function by inducing TNF-α and LTB 4 production, and on neutrophils, by stimulating chemotaxis and LTB 4 production, via cytosolic PLA 2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate in vitro anti- Leishmania amazonensis activity of a Phospholipase A 2 (Asp49-PLA 2 ) isolated from the venom of Bothrops jararacussu and its encapsulated form. Asp49-PLA 2 (2 mg/mL) was added to a lipid mixture, solubilized in chloroform and dried under nitrogen flow. The lipid vesicles were formed homogeneously using the extrusion method, physicochemically characterized by their diameters, zeta potentials, encapsulation rate and also submitted to a molecular docking in silico analysis. The activity of Asp49-liposomes was evaluated in vitro against promastigote forms of L. amazonensis and J774 macrophages. Parasite and macrophage viabilities using MTT method were assessed after 48 h incubation. L. amazonensis -infected macrophages were also incubated with encapsulated Asp49 and in solution form. The amastigote forms were counted inside the infected macrophages and the culture supernatants were collected for nitrites and TNF-α quantifications. Asp49-PLA 2 in solution form displayed an anti - Leishmania concentration-dependent effect. Asp49-liposomes were able to reduce 78% of promastigote forms and preserved 82% of J774 macrophages' viability. After 48 h of incubation with nanoencapsulated Asp49-PLA 2 there was a significant reduction in the number of amastigotes (55%; p < 0.05) compared to the control group. When the macrophages were infected and incubated with Asp49-liposomes a significant increase in the production of nitrites and TNF-α was observed when compared to infected cells alone. The results indicated that the liposomal system achieved in this study is a promising tool to enhance microbicidal activity of the infected macrophages, conferring a biotechnological therapeutic approach against experimental leishmaniasis. [ABSTRACT FROM AUTHOR]

Snake venom is a complex mixture of active compounds consisting of 80–90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A 2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A 2 homologue from the venom of Bothrops neuwiedi urutu . The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA 2 s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA 2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A 2 from Bothrops snake venoms, and identified this molecule as a novel PLA 2 isoform from B . neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa . The results allowed for the identification of a new myotoxin isoform with PLA 2 structure with promising biotechnological applications. [ABSTRACT FROM AUTHOR]

The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A 2 called MsPLA 2 -I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC 50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum , which are unprecedented for Micrurus venoms. MsPLA 2 -I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii . The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA 2 from Micrurus altirostris . This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA 2 -I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA 2 -I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential. [ABSTRACT FROM AUTHOR]

Abstract: Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA2 (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin. [Copyright &y& Elsevier]

Abstract: Phospholipases A2 are important components of snake venoms, the basic isoforms have been more extensively studied than the acidic groups, maybe due to their higher toxicity. Trying to better understand the role of the acidic isoforms on the envenomation process, an acidic phospholipase A2 was purified from Bothrops moojeni snake venom through two chromatographic steps (BmooPLA2). The enzyme showed a relative molecular mass of 13,601Da, pI 5.2, high phospholipase activity, bactericidal effect, moderate cytotoxic activity and was able to inhibit platelet aggregation. Moreover, BmooPLA2 induced moderate in vivo edema and hypotensive effect. The 414bp cDNA encoding the BmooPLA2 was cloned and expressed in Escherichia coli. The recombinant BmooPLA2 showed phospholipase and inhibitory activities on platelet aggregation similar to those of the native protein. A comparative study between BmooPLA2, the acidic (BthA-I) and basic (BthTX-II) PLA2 from B. jararacussu venom showed that the effects of BmooPLA2 and BthA-I-PLA2 are similar. BmooPLA2 is the first isolated and characterized non-myotoxic PLA2 from B. moojeni snake venom. The recombinant PLA2 can substitute the native toxin in studies aiming its biotechnological application in order to help the preservation of this endangered species. These data along with the preliminary structural studies here reported will provide a better understanding of this important class of proteins. [Copyright &y& Elsevier]

Abstract: Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. [Copyright &y& Elsevier]

Abstract: Phospholipases A2 (PLA2s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA2 inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated αBaltMIP, showed it to be a glycoprotein with Mr of ∼24,000 for the monomeric subunit. CD spectra of the PLA2/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of αBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. [Copyright &y& Elsevier]

Abstract: This work aimed at the isolation and structural/functional characterization of a phospholipase A2 (CgPLA2) from the extract of the anemone Condylactis gigantea. CgPLA2 was isolated with a high purity level through three chromatographic steps, showing pI ˜8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA2 showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca2+-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA2 with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA2 revealed 84%, 61%, 43% and 42% similarity to the PLA2s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively. [Copyright &y& Elsevier]

Abstract: Phospholipases A2 (Asp49-PLA2s) are enzymes responsible for cellular membrane disruption through Ca2+-dependent hydrolysis of phospholipids. A class of these proteins (Lys49-PLA2s) does not show catalytic activity but can exert a pronounced local myotoxic effect that is not neutralized by serum therapy. In this work, we present five structures of Lys49-PLA2s from snakes of the Bothrops genus in apo form, complexed with PEG molecules and chemically modified by p-bromofenacil bromide (BPB), a classic inhibitor of PLA2. We present herein an extensive structural analysis including: (i) the function of hydrophobic long-chain molecules as Lys49-PLA2s inhibitors, (ii) the role of Lys122, previously indicated as being responsible for Lys49-PLA2s catalytic inactivity and, (iii) a structural comparison of the Ca2+-binding loop region between Lys49 and Asp49-PLA2s. The Lys122 analysis of 30 different monomers for apo and complexed Lys49-PLA2s structures shows that this residue is very flexible and may bind to different carboxyl groups giving stability to the crystal structures. The structural comparisons of the Ca2+-binding loop region between Lys49 and Asp49-PLA2s reveal the importance of the Tyr28 residue conservation in Asp49-PLA2s to the integrity of this loop. The Tyr28 residue stabilizes this region by an interaction with Gly35 residue. In Lys49-PLA2s and low-catalytic Asp49-PLA2s this interaction does not occur, preventing the binding of Ca2+. [Copyright &y& Elsevier]

Abstract: For the first time, the structure of a catalytic inactive phospholipase A2 homolog (Lys49-PLA2s) complexed with p-bromophenacyl bromide (BPB) has been solved by X-ray crystallography. Lys49-PLA2s are among the main components of Viperidae snake venoms, causing myonecrosis and other actions despite their catalytic inactivity. BPB, a classic inhibitor of catalytic-active PLA2s, has been used since the 1970s because it binds specifically the His48 residue of the catalytic site. Curiously, when Lys49-PLA2 is chemically modified by BPB, it causes a partial inhibition of the myotoxic function which is associated with the C-terminus and not with the catalytic site. The structure of PrTX-I complexed to BPB revealed unambiguously that the inhibitor binds covalently to His48, causing a distortion of the Ca2 +-binding loop region and C-terminus rearrangement in one of its monomers. The comparison between the apo and BPB-complexed PrTX-I structures showed an increased symmetry between the two monomers with the formation of an interchain hydrogen bond between Tyr119 residues. PrTX-I undergoes tertiary and quaternary structural changes when complexed to BPB which could be related to reduction of myotoxicity and other toxic activities. We also proposed a novel myotoxic inhibition hypothesis integrating “myotoxic” and “active” sites for bothropic Lys49-PLA2s. [Copyright &y& Elsevier]

Abstract: The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115–129 C-terminal region. BPB-BthTX-I presented cytotoxicity of 10–40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer. [Copyright &y& Elsevier]

Abstract: In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5kDa, pI=7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A2 activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5–40°C. Assays with metal ions showed that Ca2+ is an activator, whereas Zn2+ and Hg2+ inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs. [Copyright &y& Elsevier]

Abstract: An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A2 from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated αBjussuMIP, showed it to be an oligomeric glycoprotein with M r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% α-helix, 18% β-sheet, 10% β-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either αBjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of αBjussuMIP with other snake inhibitors showed a sequence similarity of 73–92% with these αPLIs. αBjussuMIP was relatively stable within the pH range of 6–12 and temperatures from 0°C to 80°C, even after deglycosylation. The results showed effects against Bothrops phospholipase A2 activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that αBjussuMIP may prove useful in the treatment of snakebite envenomations. [Copyright &y& Elsevier]

Abstract: This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M r ∼14,000 for the monomer and 28,000Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose–time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115–129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. [Copyright &y& Elsevier]

Abstract: A myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu) and other Asp49-PLA2s. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA2s, making a hydrogen bond with the atom Oδ2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA2s which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA2s indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA2s. [Copyright &y& Elsevier]

Abstract: This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA2, a new myotoxic acidic phospholipase A2 from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA2 is a single-chain protein of 15.8kDa and pI 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA2s from snake venoms. Its specific activity was 585.3U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA2 with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA2 induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24h after injection. Acidic myotoxic PLA2s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities. [Copyright &y& Elsevier]

Abstract: MjTX-II, a myotoxic phospholipase A2 (PLA2) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA2s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others “bioactive sites” distinct from the catalytic site in snake venom myotoxic PLA2s. [Copyright &y& Elsevier]

An acidic phospholipase A2 (PLA2) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: α-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-Å resolution, respectively, for the complexes with α-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred. [Copyright &y& Elsevier]

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A 2 (PLA 2 s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA 2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA 2 s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA 2 s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA 2 -like. Acidic PLA 2 s catalyzed the degradation of all substrates evaluated; however, for the basic PLA 2 s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA 2 s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum , while the basic PLA 2 s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA 2 and Lys49-PLA 2 were associated with one another, denoting synergistic action between these PLA 2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA 2 s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents. [Display omitted] • Nine PLA 2 s isoforms were isolated from B. asper venom. • Four acidic and five basic isoforms of PLA 2 s were characterized. • Five of the isolated PLA 2 s represent unpublished isoforms. • The basic isoforms of PLA 2 s have an antiparasitic effect against P. falciparum. • Synergism between basic isoforms of PLA 2 s increases antiplasmodial effect. [ABSTRACT FROM AUTHOR]