Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach

Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains a and P. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family. (C)0 2007 Elsevier Ltd. All rights reserved.

ACUTE KIDNEY INJURY CAUSED BY Crotalus AND Bothrops SNAKE VENOM: A REVIEW OF EPIDEMIOLOGY, CLINICAL MANIFESTATIONS AND TREATMENT

SUMMARY Ophidic accidents are an important public health problem due to their incidence, morbidity and mortality. An increasing number of cases have been registered in Brazil in the last few years. Several studies point to the importance of knowing the clinical complications and adequate approach in these accidents. However, knowledge about the risk factors is not enough and there are an increasing number of deaths due to these accidents in Brazil. In this context, acute kidney injury (AKI) appears as one of the main causes of death and consequences for these victims, which are mainly young males working in rural areas. Snakes of the Bothrops and Crotalus genera are the main responsible for renal involvement in ophidic accidents in South America. The present study is a literature review of AKI caused by Bothrops and Crotalus snake venom regarding diverse characteristics, emphasizing the most appropriate therapeutic approach for these cases. Recent studies have been carried out searching for complementary therapies for the treatment of ophidic accidents, including the use of lipoic acid, simvastatin and allopurinol. Some plants, such as Apocynaceae, Lamiaceae and Rubiaceae seem to have a beneficial role in the treatment of this type of envenomation. Future studies will certainly find new therapeutic measures for ophidic accidents.

Effect of perimuscular injection of Bothrops jararacussu venom on plasma creatine kinase levels in mice: influence of dose and volume

The effect of dose and volume of a perimuscular injection of Bothrops jararacussu venom on myonecrosis of skeletal muscle was studied in mice. An increase of the venom dose (0.25 to 2.0 µg/g) at a given volume (50 µl) resulted in an increase in plasma creatine kinase (CK) levels 2 h after injection. Plasma CK activity increased from the basal level of 129.27 ± 11.83 (N = 20) to 2392.80 ± 709.43 IU/l (N = 4) for the 1.0 µg/g dose. Histological analysis of extensor digitorum longus muscle 4 h after injection showed lesion of peripheral muscle fibers, disorganization of the bundles or the complete degeneration of muscle fibers. These lesions were more extensive when higher doses were injected. Furthermore, an increase in volume (12.5 to 100 µl) by dilution of a given dose (0.5 µg/g) also increased plasma CK levels from 482.31 ± 122.79 to 919.07 ± 133.33 IU/l (N = 4), respectively. These results indicate that care should be taken to standardize volumes and sites of venom injections.

The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 µg/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 µg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 µM acetylcholine alone and cumulative concentrations of 1 µM to 10 mM were unaffected. At venom concentrations higher than 50 µg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 µg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA(2)) and II (Asp49 PLA(2)) and their possible blockage by indomethacin. BthTX-1 (5 mu g/ml) and BthTX-II (5 mu g/ml) increased perfusion pressure (PP; ct(120) = 110.28+/-3.70 mmHg; BthTX I = 171.28+/-6.30* mmHg; BthTX II = 175.50+/-7.20* mmHg), renal vascular resistance (RVR; ct(120) = 5.49+/-0.54 mmHg/ml.g(-1) min(-1); BthTX I = 8.62+/-0.37* mmHg/ml g(-1) min(-1); BthTX II=8.9+/-0.36* mmHg/ml g(-1) min(-1)), urinary flow (UF; ct(120)= 0.14+/-0.01 ml g(-1) min(-1); BthTX I=0.32+/-0.05* ml g(-1) min(-1); BthTX II=0.37+/-0.01* ml g(-1) min(-1)) and glomerular filtration rate (GFR; ct(120)=0.72+/-0.10 ml g(-1) min(-1); BthTX I=0.85+/-0.13* ml g(-1) min(-1); BthTX II=1.22+/-0.28* ml g(-1) min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa+; ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK+;ct(120)=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA(2). In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA(2). (C) 2005 Elsevier Ltd. All rights reserved.

A Catalytically Inactive Lys49 PLA2 Isoform from Bothrops jararacussu venom that Stimulates Insulin Secretion in Pancreatic Beta Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA(2) activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 mug/ml) plus AE (400 mug/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 mug/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 mug/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 mug/ml, I h) did not reveal any change in muscle fibers, but severe necrosis induced by -BV or toxins (20 mug/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA(2), activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 mug of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 mug), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with I h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors. (C) 2004 Elsevier GmbH. All rights reserved.

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) 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-PLA(2)S 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 PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis Venom

BnSP-7, a Lys49 myotoxic phospholipase A, homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues, the cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pi and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A, homologues from other Bothrops sp, venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick, biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes, Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophhenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr), the bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide, the neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide, the rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca2+-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II. (C) 2000 Academic Press.

Crystallization and preliminary X-ray diffraction analysis of a myotoxic Lys49-PLA(2) from Bothrops jararacussu venom complexed with p-bromophenacyl bromide

For the first time, a non-catalytic and myotoxic Lys49-PLA(2) (BthTX-I from Bothrops jararacussu venom) has been crystallized with BPB inhibitor. X-ray diffraction data were collected and electron-density calculations showed that the ligand is bound to the His48 residue. BthTX-I with His48 chemically modified by BPB shows strongly reduced myotoxic and cytotoxic activities. This suggests a biological correlation between the modification of His48, which is associated with catalytic activity of PLA(2)s, and other toxicological activities of Lys49-PLA(2)s.

Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A(2) with low catalytic activity from Bothrops jararacussu venom

For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A(2) (Asp49-PLA(2)) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA(2) PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA(2)s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA(2) from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA(2)s.

Crystallization and preliminary X-ray analysis of S-III-SPIII, a phospholipase A2 isolated from the venom of Bothrops jararacussu.

Large single crystals have been obtained of S-III-SPIII, a phospholipase A(2) from the venom of Bothrops jararacussu. The crystals belong to the orthorhombic system space group C222, and diffract X-rays to a resolution of 1.9 Angstrom. Preliminary analysis reveals the presence of one molecule in the crystallographic asymmetric unit. The crystal structure is currently being determined using molecular replacement techniques.