Phtx-ii A Basic Myotoxic Phospholipase A2 From Porthidium Hyoprora Snake Venom, Pharmacological Characterization And Amino Acid Sequence By Mass Spectrometry.

A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.

Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxic phospholipase A(2) from Bothrops neuwiedi venom

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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.

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.

Comparative biochemical studies of myotoxic phospholipase A(2) from Bothrops venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA(2)-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.

Crystal structure of piratoxin-I: A calcium-independent, myotoxic phospholipase A(2)-homologue from Bothrops pirajai venom

The crystal structure of Piratoxin-I (PrTX-I) a Lys49 homologue isolated from the venom of Bothrops pirajai has been determined and refined at 2.8 Angstrom to a crystallographic residual of 19.7% (R-free = 29.7%). Amino-acid sequence differences between catalytically active phospholipases and PrTX-I in the putative Ca2+-binding loop, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered conformation of this loop, the analysis of the position of the E-amino group of Lys49 in the PrTX-I structure indicates that it fills the site normally occupied by the calcium ion in the catalytically active phospholipases, In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus (App), PrTX-I is present as a dimer in the crystalline state, as observed in the structures of myotoxin II from Bothrops asper and Bothropstoxin I from Bothrops jararacussu. The two molecules in the asymmetric unit in the crystal structure of PrTX-I are related by a nearly perfect two-fold symmetry axis, yet the dimeric structure is radically different from the dimeric structure of the phospholipase from Crotalus atrox. In the C. atrox structure the dimer interface occludes the active sites, whereas in the PrTX-I structure they are exposed to solvent, (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Crystal structure of myotoxin-II: A myotoxic phospholipase A2 - Homologue from Bothrops moojeni venom.

The crystal structure of Myotoxin-II (MjTX-II), a Lys49 PLA(2)-homologue from Bothrops moojeni venom has been determined and refined at 2.0 Angstrom to a crystallographic residual of 19.7% (R-free = 28.1%). MjTX-II is a dimer in the crystal, with the monomers in the asymmetric unit related by a two-fold symmetry axis running through the dimer interface. The dimers of MjTX-II and the Lys49 PLA(2) from B. asper venom are similar, however the relative orientations of the monomers suggests a flexible dimer interface, which serves as a hinge between the two molecules.

A structure based model for liposome disruption and the role of catalytic activity in myotoxic phospholipase A(2)S

Venom phospholipase A(2)s (PLA(2)s) display a wide spectrum of pharmacological activities and, based on the wealth of biochemical and structural data currently available for PLA(2)S, mechanistic models can now be inferred to account for some of these activities. A structural model is presented for the role played by the distribution of surface electrostatic potential in the ability of myotoxic D49/K49 PLA(2)s to disrupt multilamellar vesicles containing negatively charged natural and non-hydrolyzable phospholipids. Structural evidence is provided for the ability of K49 PLA(2)s to bind phospholipid analogues and for the existence of catalytic activity in K49 PLA(2)s. The importance of the existence of catalytic activity of D49 and K49 PLA(2)s in myotoxicity is presented. (C) 2003 Elsevier Ltd. All rights reserved.

Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxic phospholipase A(2) from Bothrops neuwiedi venom

The LY549-PLA(2)s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA(2) is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA(2) was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)(12)COOH) and its overall structure was refined at 2.2 angstrom resolution. The Bn IV crystals belong to monoclinic space group P2(1) and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 angstrom. The biological assembly is a "conventional dimer" and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Amino acid sequence of a myotoxic Lys49-phospholipase A(2) homologue from the venom of Cerrophidion (Bothrops) godmani

The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A( 2 )(PLA(2)) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA, showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca2+, since the residues involved in forming the catalytic network of PLA(2)s (His-48, Tyr-52 and Asp-99) an conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA(2)s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA(2). The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca2+-independent membrane damaging activity. (C) 1998 Elsevier B.V. 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.

Crystallographic and spectroscopic characterization of a molecular hinge: Conformational changes in bothropstoxin I, a dimeric Lys49 phospholipase A2 homologue

Bothropstoxin I(BthTX-I) from the venom of Bothrops jararacussu is a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49-->Lys substitution, disrupts the integrity of lipid membranes by a Ca2+-independent mechanism, the crystal structures of two dimeric farms of BthLTX-I which diffract X-rays eo resolutions of 3.1 and 2.1 Angstrom have been determined, the monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal alpha-helical regions and the tips of the beta-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in open and closed dimer conformations, Spectroscopic Investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface, the possible relevance of this structural transition in the Ca2+-independent membrane damaging activity is discussed. (C) 1998 Wiley-Liss, Inc.

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as delta lesions were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. © 1991.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes. (C) 2009 Elsevier Ltd. All rights reserved.