Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Isolation and Characterization of a Serine Protease,Ba III-4, from Peruvian <Emphasis Type="Italic">Bothrops atrox</Emphasis> venom

A serine protease from Bothrops atrox (Peruvian specimen’s venom) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. This protein was denominated Ba III-4 (33,080.265 Da determinated by MALDI-TOF mass spectrometry) and showed pI of 5.06, Km 0.2 × 10⁻¹ M and the V _máx 4.1 × 10⁻¹ nmoles p-NA/lt/min on the synthetic substrate BapNA. Ba III-4 also showed ability to coagulate bovine fibrinogen. The serine protease was inhibited by soyben trypsin inhibitor and DA2II, which is an anti-hemorrhagic factor isolated from the opossum specie Didelphis albiventris. The primary structure of Ba III-4 showed the presence of His(44), Asp(94) and Ser(193) residues in the corresponding positions to the catalytic triad established in the serine proteases and Ser(193) are inhibited by phenylmethylsulfonylfluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro, as well as 12 half-cysteine residues. Ba III-4 contained 293 amino acid residues and the primary structure of VIGGDECDIN EHPFLAFMYY SPRYFCGMTL INQEWVLTAA HCRYFCGMTL IHLGVHRESE KANYDEVRRF PKEKYFIFCD NNFTDDEVDK DIMLIRLDKP VSNSEHIAPL SLPSNPPSVG SVCRIMGWGQ TTTSPIDVLS PDEPHCANIN LFDNTVCHTA HPQVANTRTS TDTLCAGDLQ GGRDTCNGDS GGPLICNEQL HGILSWGGDP CAQPNKPAFY TKVYYFDHPW IKSIIAGNKK TVNFTCPPLR SDAKDDSTTY INQEWDWVLT AEHCDRTHMR NSFYDYSSIN SDS. Titration experiments did not show the presence of free sulfhydryl groups after 4 h incubation, nor were differences found in relation to titration kinetics in the presence of nondenaturating buffer. The isolation of this protein, Ba III-4, is of potential interest for the understanding of the pathomechanism of the snake venom action and for the identification of new blood coagulation enzymes of natural sources.     

Neurotoxic,Myotoxic and Cytolytic Activities of the New Basic PLA<Subscript>2</Subscript> Isoforms BmjeTX-I and BmjeTX-II Isolated from the <Emphasis Type="Italic">Bothrops marajoensis</Emphasis> (Marajó Lancehead) Snake Venom

The BmjeTX-I and BmjeTX-II isoforms of PLA₂ were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS–PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA₂ show high degree of homology with basic PLA₂ myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In chick preparations, contractures to exogenous acetylcholine (55 and 110 μM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA₂. These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA₂ also induced moderate marked paw edema, evidencing the local increase in vascular permeability. Since both isoforms of PLA₂ exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes.     

Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom

This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes.     

Structural Characterization and Neuromuscular Activity of a New Lys49 Phospholipase A2 Homologous (Bp-12) Isolated from Bothrops pauloensis Snake Venom

Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on μ-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA₂ from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 ± 6 min (1.4 μM) and 16 ± 6 min (3.6 μM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca²⁺ is replaced by Sr²⁺, being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA₂ family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.     

Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma

A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.     

Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom

A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40 degrees C. Full PLA2 activity required Ca2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+. The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu2+ also reduced the enzymatic activity. Bj V had PLA2 activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.     

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.     

Involvement of cyclodipeptides in the competition of bacterial communities in the oligotrophic Churince aquatic system of Cuatro Ciénegas Basin dominated by Gammaproteobacteria

The Cuatro Ciénegas Basin (CCB) within the Chihuahuan Desert in México is an extremely oligotrophic oasis with negligible phosphorous levels, described as a hot spot of biodiversity, not only in stromatolites and microbial mats, but also in living forms in general. The microorganisms possess the capability to produce a wide variety of virulence factors, antibiotics, and quorum-sensing (QS) crosstalk signals such as non-ribosomal cyclodipeptides (CDPs) which enables them to colonize diverse ecological niches. In the aquatic system of CCB known as Churince, a bacterial population was isolated from the Lagunita pond dominated by Gammaproteobacteria. In this work, we determined the relationships between the antagonism and CDPs production in this bacterial population. Results indicate that 68% of isolates showed antagonistic effects over other isolates, correlating with production of CDPs and the antibiotic 2,4-diacetylphloroglucinol (DAPG). Although a minority of the isolates were capable of inducing a QS biosensor strain, bacterial QS interference was not the main mechanism in the antagonism observed. Thus, our results indicate that CDPs primarily, and DAPG to a lesser degree, are involved with the growth-inhibition competition mechanisms of bacterial communities in the Lagunita pond and was associated with a Gammaproteobacteria dominancy phenomena.