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S L Da Silva,
C A Dias-Junior,
P A Baldasso,
D C S Damico,
B M A Carvalho,
A Garanto,
G Acosta,
E Oliveira,
F Albericio, A M Soares,
S Marangoni,
R R Resende
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ABSTRACT: Crotalus oreganus abyssus is a rattlesnake that is usually found in the Grand Canyon, United States of America. Knowledge regarding the composition of C. o. abyssus venom is scarce. New natriuretic peptides (NPs) have been isolated and characterized from the venoms of members of the Crotalinae family. The NP family comprises three members, ANP (atrial natriuretic peptide), BNP (b-type natriuretic peptide) and CNP (c-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. The aim of the present study was to characterize a novel natriuretic-like peptide (Coa_NP2), isolated from C. o. abyssus venom. The Coa_NP2 presents an average molecular mass of 3419.88Da (theoretical average molecular mass 3418.94Da, monoisotopic molecular mass 3416.66Da and theoretical PI 7.78) and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides. The peptide has 32 amino acids and its complete sequence is SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. Coa_NP2 is a natriuretic peptide of the ANP/BNP-like family, since the carboxyterminal region of CNP has its own NP domain. We demonstrate, herein, that Coa_NP2 produces a dose-dependent decrease in mean arterial pressure in rats, followed by significant increases in concentrations of markers of nitric oxide formation measured in the plasma and vasorelaxation in a thoracic aortic ring bath. The structural and biological aspects confirm Coa_NP2 as a new natriuretic peptide, isolated from snake venom.
Peptides 05/2012; 36(2):206-12. · 2.43 Impact Factor
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ABSTRACT: This study investigated the effect of a pool of peptides, isolated from venom of Crotalus durissus terrificus (South American rattlesnake) on glucose concentration in C57BL/6 mice fed on a high-fat diet for 6weeks. The pool of peptides
(molecular mass around of 10kDa) was obtained using a MidJet apparatus with a cartridge of 10KDa. The peptide pool was injected
intraperitoneally in mice in a single dose (0.5mg/animal) or multiple doses (0.2mg/dose). After predetermined times (30,
60, 90 and 120min) post injections, venous blood samples were collected for enzymatic measurement of serum glucose using
a commercial glucose kit (glucose oxidase method). High-fat fed mice showed an increase in blood glucose concentration, in
comparison with mice fed on the chow diet. Thirty minutes after a single dose of the peptide pool, high-fat fed animals showed
a significant decrease (~47%) in glycemia. However, the glucose level increased again at 60 and 120min. Conversely, after
multiple injections of the pool of peptides administered every 30min, the blood glucose concentration in the high-fat mice
was significantly decreased (~37%) and remained at low levels until 120min. These results suggest that the tested pool of
peptides from Crotalus durissus terrificus contained a peptide (or peptides) with a beneficial role on glucose-lowering action of high-fat fed mice.
KeywordsBioactive peptides–
Crotalus durissus terrificus
–Snake venom–Hyperglycemia–Glucose
International Journal of Peptide Research and Therapeutics 04/2012; 17(3):225-230. · 0.99 Impact Factor
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S. L. Da Silva,
J. R. Almeida,
L. M. Resende,
W. Martins,
F. A. F. A. Henriques,
P. A. Baldasso, A. M. Soares,
A. G. Taranto,
R. R. Resende,
S. Marangoni,
C. A. Dias-Junior
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ABSTRACT: Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A
good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting
the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide)
and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study,
we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated
from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98Da and its amino acid sequence presents the loop region that
is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic
peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments
showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease
in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings
that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide
isolated from snake venom, thus expanding the diversification of venom components.
KeywordsNatriuretic peptide–
Crotalus oreganus abyssus
–Bioactive peptides–Nitric oxide
International Journal of Peptide Research and Therapeutics 04/2012; 17(3):165-173. · 0.99 Impact Factor
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E S de Alvarenga,
S A Silva,
L C A Barosa,
A J Demuner,
A G Parreira,
R I M A Ribeiro,
S Marcussi,
J M S Ferreira,
R R Resende,
P A Granjeiro,
J A Silva, A M Soares,
S Marangoni,
S L Da Silva
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ABSTRACT: Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A(2) (PLA(2)) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA(2) edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K(I)) for Lac01 and Lac02 were approximately 740 μM, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 μM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA(2) in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA(2) (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA(2).
Toxicon 10/2010; 57(1):100-8. · 2.51 Impact Factor
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ABSTRACT: Crotoxin B is a basic phospholipase A(2) found in the venom of several Crotalus durissus ssp. rattlesnakes and is one of the subunits that constitute crotoxin, the main component of the venom of these snakes. This heterodimeric toxin is related to important envenomation effects such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, the first structural data only became available in 2007 (for crotoxin B from C. durissus terrificus) and showed an ambiguous result for the biological assembly, which could be either dimeric or tetrameric. In this work, the crystallization, X-ray diffraction data collection at 2.2 A resolution and molecular-replacement solution of a dimeric complex formed by two crotoxin B isoforms from C. durissus collilineatus venom is presented.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2009; 65(Pt 10):1011-3. · 0.51 Impact Factor
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ABSTRACT: Crotoxin B is a basic phospholipase A2 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 A resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2008; 63(Pt 12):1067-9. · 0.51 Impact Factor
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ABSTRACT: In this work, the inhibitory profile of anti-myotoxic factor (BaMIP) derived from the blood of the snake Bothrops asper (“terciopelo”), known to neutralize the basic phospholipase A2 myotoxins from its own venom, was determined.
Neutralization of the enzymatic activity of PLA2s from different structural groups (I, II, and III) was determined using three different methods.
Furthermore, in order to establish the pharmacological potential of this inhibitor against the toxic properties of PLA2s from different venom sources, a panel of neutralization studies for the following toxic properties were performed: myotoxic activity, anticoagulant effect, edema and intraventricular lethality.
Results indicate that BaMIP is highly specific for PLA2s towards group II and inhibits most of its PLA2s activity. On the other hand, its inhibitory effect against group I and III PLA2s is minimal, which correlates with the results of enzymatic activity.
It was also determined that BaMIP particularly inhibits the toxic properties of group II PLA2s, but not that of group I and III PLA2s, which also correlate with the enzymatic results. The myotoxic effect as well as the edematogenic, anticoagulant, and lethal properties were all neutralized in group II PLA2s. In contrast to the enzyme results, the intraventricular lethality test indicated that BaMIP also neutralizes the bee venom group III PLA2.
Even though the variation observed in the specificity and intensity in the inhibition of the enzymatic activity and of the toxic effects associated with PLA2s could be explained by differences in the affinity of the BaMIP (inhibitor) by the PLA2s and by differences in the sensibility and kinetic conditions of the used methods, respectively, this worked did not intend to determine the constant of affinity between the BaMIP and the PLA2s included in the study, even though the results reflect it. It is important to indicate that yet when the enzymatic activity may be a factor that contributes to the development of some toxic effects produced by these proteins type PLA2s, it may not be the only factor involved in toxicity.
J. Venom. Anim. Toxins incl. Trop. Dis. 01/2007; 13(1):315.
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ABSTRACT: For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A2 (Asp49-PLA2) 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-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA2s.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2006; 62(Pt 8):765-7. · 0.51 Impact Factor
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ABSTRACT: For the first time, a non-catalytic and myotoxic Lys49-PLA2 (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 PLA2s, and other toxicological activities of Lys49-PLA2s.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2006; 62(Pt 6):600-3. · 0.51 Impact Factor
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ABSTRACT: A new myotoxic Lys49-phospholipase A2 isolated from Bothrops moojeni snake venom has been crystallized. The crystals diffracted to 2.18 A resolution using a synchrotron-radiation source and belong to space group C2. The unit-cell parameters are a = 56.8, b = 125.0, c = 64.7 A, beta = 105.5 degrees. Preliminary analysis indicates the presence of four molecules in the asymmetric unit. This may suggest a new quaternary structure for this Lys49-phospholipase A2 in contrast to the dimeric and monomeric structures solved so far for this class of proteins.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2005; 61(Pt 10):882-4. · 0.51 Impact Factor
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ABSTRACT: Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.
Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology 08/2004; 138(4):429-36. · 2.62 Impact Factor
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ABSTRACT: In order to produce explants of Mandevilla illustris (Vell) Woodson for the "Cerrado in vitro", the Germplasm Bank of UNAERP, we carried out a micropropagation protocol using MS or MS/3 medium supplemented with different concentrations of 6-benzyladeninepurine (BA), Zeatin or 2-isopentenyladenine for nodal segment growth, and alpha-naphthaleneacetic acid, indole-3-butyric acid (IBA) or 1,4 dithiothreitol for rooting. For nodal segments, all the cytokinins tested yielded similar results. However, 2.22 micro M BA is more economical to use. MS/3 medium supplemented with 0.49 micro M IBA was the most appropriate medium for rooting, resulting in 29% rooted explants. The crude aqueous extract from the subterranean system (SS) of M. illustris was assayed for its inhibitory action on the enzymatic activity of Crotalus durissus terrificus snake venom, isolated basic phospholipase A2 (CB) and crotoxin. It totally inhibited the phospholipase activity of crude Cdt venom and CB toxin and inhibited the phospholipase activity of crotoxin by 49%. The toxic action of both the crude venom and crotoxin was partially inhibited-there was a prolonged survival time and a 40.0% decrease in lethality.
Plant Cell Reports 04/2004; 22(8):549-52. · 2.27 Impact Factor
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ABSTRACT: In order to produce explants of Mandevilla illustris (Vell) Woodson for the Cerrado in vitro, the Germplasm Bank of UNAERP, we carried out a micropropagation protocol using MS or MS/3 medium supplemented with different concentrations of 6-benzyladeninepurine (BA), Zeatin or 2-isopentenyladenine for nodal segment growth, and -naphthaleneacetic acid, indole-3-butyric acid (IBA) or 1,4 dithiothreitol for rooting. For nodal segments, all the cytokinins tested yielded similar results. However, 2.22M BA is more economical to use. MS/3 medium supplemented with 0.49M IBA was the most appropriate medium for rooting, resulting in 29% rooted explants. The crude aqueous extract from the subterranean system (SS) of M. illustris was assayed for its inhibitory action on the enzymatic activity of Crotalus durissus terrificus snake venom, isolated basic phospholipase A2 (CB) and crotoxin. It totally inhibited the phospholipase activity of crude Cdt venom and CB toxin and inhibited the phospholipase activity of crotoxin by 49%. The toxic action of both the crude venom and crotoxin was partially inhibited—there was a prolonged survival time and a 40.0% decrease in lethality.
Plant Cell Reports 01/2004; 22(8):549-552. · 2.27 Impact Factor
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ABSTRACT: Lys49-Phospholipase A2 (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region permits quaternary structural transitions between "open" and "closed" membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78A, (ii) MjTX-II/STE complex at a resolution of 1.8 A and (iii) BthTX-I/DMPC complex at 2.72A. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (iii) and using using a Synchrotron Radiation Source (Laboratório Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).
Protein and Peptide Letters 11/2003; 10(5):525-30. · 1.94 Impact Factor
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ABSTRACT: Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The alpha-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.
Toxicon 01/2002; 39(12):1863-9. · 2.51 Impact Factor
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ABSTRACT: Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum (Didelphis albiventris) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point < 4. The two subunits were separated by ion-exchange HPLC. The N-terminal sequences of both subunits (LKAMDPTPXLWIETESP, where X is Arg-9 and Pro-9, respectively) showed a high degree of identity with other serum inhibitors isolated from different marsupials. Functional studies pointed out that ABC inhibits the hemorrhagic and proteolytic activities on fibrin, fibrinogen, and casein induced by the metalloproteases BaP1 and BaH4 isolated from B. asper venom. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A(2) isolated from the same venom. Moreover, it had inhibitory effects on the phospholipase A(2) activity of the crude venom as well as the isolated venom phospholipases A(2).
Biochemical Pharmacology 12/2001; 62(11):1521-9. · 4.70 Impact Factor
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ABSTRACT: Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).
The International Journal of Biochemistry & Cell Biology 10/2001; 33(9):877-88. · 4.63 Impact Factor
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ABSTRACT: The pathological alterations induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi, were studied in mice. Neuwiedase was devoid of hemorrhagic activity when tested in the skin up to a dose of 200 microgram, and also after intramuscular injection in the gastrocnemius. However, it induced bleeding when applied onto the mouse cremaster muscle in intravital microscopy experiments, and caused pulmonary hemorrhage when injected intravenously at doses higher than 5 microgram/g. Median lethal dose (LD(50)) by the intravenous route was 5 microgram/g, whereas LD(50) of crude venom was 0.47 microgram/g. After intramuscular injection, neuwiedase induced a mild myotoxic effect, evidenced histologically and by the increment in plasma creatine kinase activity, but it was devoid of hemorrhagic and thrombotic effects. In contrast, crude B. neuwiedi venom induced prominent hemorrhage and myonecrosis in gastrocnemius muscle. Both venom and neuwiedase induced an inflammatory reaction in muscle tissue characterized by abundant polymorphonuclear leukocytes. Moreover, a conspicuous edema developed in the foot pad after subcutaneous injection of neuwiedase. Anti-neuwiedase antibodies produced in rabbits were effective in the neutralization of hemorrhagic activity of crude venom, evidencing immunological cross-reactivity between neuwiedase and other hemorrhagic metalloproteinases present in the venom, and suggesting that metalloproteinases devoid of, or having low, hemorrhagic activity could be good immunogens to generate antibodies effective against high molecular mass metalloproteinasas having potent hemorrhagic activity. It is concluded that neuwiedase, despite its lack of hemorrhagic effect when injected in the gastrocnemius muscle, contributes to local tissue damage by inducing edema, inflammatory infiltrate and mild myotoxicity, and by degrading extracellular matrix components. In addition, large doses of neuwiedase may contribute to pulmonary bleeding
Biochimie 07/2001; 83(6):471-9. · 3.02 Impact Factor
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ABSTRACT: Myotoxic class II phospholipases A(2) from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115-129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1-15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A(2), showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins (Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A(2), regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.
Toxicon 07/2001; 39(6):879-87. · 2.51 Impact Factor
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ABSTRACT: Piratoxins (PrTX) I and III are phospholipases A2 (PLA2s) or PLA2 homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA2, while PrTX-I is a Lys49 PLA2 homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities.
Archives of Biochemistry and Biophysics 04/2001; 387(2):188-96. · 2.93 Impact Factor
