Crystal structure of myotoxin II, a monomeric Lys49-Phospholipase A(2) homologue isolated from the venom of Cerrophidion (Bothrops) godmani

Lys49-Phospholipase A(2) (Lys49-PLA(2)) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. With the aim of determining the structural basis for this novel activity, we have solved the crystal structure of myotoxin-II, a Lys49-PLA(2) isolated from the venom of Cerrophidion (Bothrops) godmani (godMT-II) at 2.8 Angstrom resolution by molecular replacement. The final model has been refined to a final crystallografic residual (R-factor) of 18.8% (R-free = 28.2%), with excellent stereochemistry. godMT-II is also monomeric in the crystalline state, and small-angle X-ray scattering results demonstrate that the protein is monomeric in solution under fisicochemical conditions similar to those used in the crystallographic studies. (C) 1999 Academic Press.

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.

Molecular characterization and phylogenetic analysis of JussuMP-I: A RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation. including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I. a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-1 metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases. (C) 2006 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of a myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom

Crystals of a myotoxic phospholipase A(2) from Bothrops neuwiedi pauloensis have been obtained. They diffracted at 2.5 Angstrom resolution using a synchrotron radiation source and belong to space group P3(1)21. Preliminary analysis shows that there are two molecules in the asymmetric unit. (C) 1999 Elsevier B.V. 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.

Rosmarinic acid, a new snake venom phospholipase A(2) inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A(2) homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea ('baleeira', 'whaler') and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA(2)s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA(2) activity of BthTX-II and, still less, the PLA(2) and edema-inducing activities of the acidic isoform BthA-1-PLA(2), from the same venom, showing therefore a higher inhibitory activity upon basic PLA(2)s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA(2)s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA(2)s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA(2). A possible model for the interaction of rosmarinic acid with Lys49-PLA(2) BthTX-I is proposed. (c) 2005 Elsevier Ltd. All rights reserved.

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.

Molecular and functional characterization of a new non-hemorrhagic metalloprotease from Bothrops jararacussu snake venom with antiplatelet activity

BjussuMP-II is an acidic low molecular weight metalloprotease (Mr similar to 24,000 and pI similar to 6.5), isolated from Bothrops jararacussu snake venom. The chromatographic profile in RP-HPLC and its N-terminal sequence confirmed its high purity level. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteases showed a high structural similarity, mainly among class P-I proteases. The molecular modeling analysis of BjussuMP-II showed also conserved structural features with other SVMPs. BjussuMP-II did not induce hemorrhage, myotoxicity and lethality, but displayed dose-dependent proteolytic activity on fibrinogen, collagen, fibrin, casein and gelatin, keeping stable at different pHs, temperatures and presence of several divalent ions. BjussuMP-II did not show any clotting or anticoagulant activity on human citrated plasma, in contrast to its inhibitory effects on platelet aggregation. The aspects broached, in this work, provide data on the relationship between structure and function, in order to better understand the effects elicited by snake venom metalloproteases. (c) 2007 Elsevier B.V. All rights reserved.

Preliminary X-ray crystallographic studies of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

Crotoxin B is a basic phospholipase A(2) found in the venom of Crotalus durissus terrificus and is one of the subunits that constitute crotoxin. This heterodimeric toxin, which is the main component of C. d. terrificus venom, is completed by an acidic, nontoxic and non-enzymatic component (crotoxin A) and is involved in important envenomation effects, such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, no crystal structure is currently available for crotoxin, crotoxin A or crotoxin B. In this work, the crystallization, X-ray diffraction data collection to 2.28 angstrom resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.

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.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) 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 similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) 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-PLA(2) 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 PLA(2), 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 PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Snake venom phospholipase A(2) inhibitors: Medicinal chemistry and therapeutic potential

Phospholipases A(2) (PLA(2)s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA(2)s. These inhibitors act on PLA(2)S through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA(2)s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA(2) inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A(2) from Bothrops jararacussu Venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization and preliminary X-ray diffraction studies of BnSP-6, a myotoxic phospholipase A2 from Bothrops neuwiedi venom

BnSP-6 (myotoxin I) is a phospholipase A2 homologue isolated from Bothrops neuwiedi pauloensis venom. Crystals of BnSP-6 were obtained which diffracted X-rays to 2.5 Angstrom resolution using a synchrotron radiation source at room temperature and belong to space group P3(1)21. The unit cell dimensions are a=b=57.7, c=131.1 Angstrom. The structure was solved by molecular replacement using the coordinates of bothropstoxin I from B. jararacussu venom. There are two molecules in the asymmetric unit.