Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Proteomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom: evidence of a broad arsenal of toxins

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Biological and structural characterization of a new PLA(2) from the Crotalus durissus collilineatus venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A(2) (PLA(2)) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA(2)s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA(2)s such as from the Bothrops snake venom. Also, this PLA(2) induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and rho-BPB showed a similar reduction. The rho-BPB did not reduce significantly the myotoxic activity induced by the PLA(2), but the anhydrous acetic acid treatment and the pre-incu-bation of PLA(2) with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA(2) with rho-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or rho-wing region may be involved in the binding of this PLA(2) to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Biological and structural characterization of a new PLA(2) from the Crotalus durissus collilineatus venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A(2) (PLA(2)) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA(2)s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA(2)s such as from the Bothrops snake venom. Also, this PLA(2) induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and rho-BPB showed a similar reduction. The rho-BPB did not reduce significantly the myotoxic activity induced by the PLA(2), but the anhydrous acetic acid treatment and the pre-incu-bation of PLA(2) with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA(2) with rho-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or rho-wing region may be involved in the binding of this PLA(2) to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Individual Venom Profiling of Crotalus durissus terrificus Specimens from a Geographically Limited Region: Crotamine Assessment and Captivity Evaluation on the Biological Activities

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Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEV-GEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the E beta-chain, and shows no effects on the gamma-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and beta-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 degrees C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 degrees C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity. (C) 2012 Elsevier Ltd. All rights reserved.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Renal and vascular effects of the natriuretic peptide isolated from Crotalus durissus cascavella venom

Crotalus durissus cascavella is a snake that is usually found in the scrublands of northeast Brazil. The components of its venom may have effects on the vascular and renal systems. Recently, a new bradykinin inhibitory peptide has been identified in the venom of the Crotalinae family. The aim of the present study was to investigate the renal and vascular effects of the natriuretic peptide isolated from the venom of Crotalus durissus cascavella (NP2_Casca). The chromatographic profile showed the fractionation of substances identified as convulxin, gyroxin, crotoxin and crotamine, as well as fractions V and VI. The electrophoretic profile of fraction V consisted of several bands ranging from approximately 6 kDa to 13 kDa, while fraction VI showed only two main electrophoretic bands with molecular weights of approximately 6 and 14 kDa. Reverse-phase chromatography showed that NP2_Casca corresponds to about 18% of fraction VI and that this fraction is the main natriuretic peptide. NP2_Casca was compared to other natriuretic peptides from other sources of snake venom. All amino acid sequences that were compared showed a consensus region of XGCFGX, XLDRIX and XSGLGCX. The group treated with NP2-Casca showed an increase in perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. The percent of total and proximal tubular transport of sodium was reduced significantly after administration of the peptide. The mean arterial pressure showed a dose-dependent decrease after infusion of NP2_Casca, and an increase in nitrite production. In the aortic ring assay, NP2_Casca caused a relaxant effect in endothelium-intact thoracic aortic rings precontracted with phenylephrine in the presence and absence of isatin. NP2_Casca failed to relax the aortic rings precontracted with an isosmotic potassium Krebs-Henseleit solution. In conclusion, the natriuretic peptide isolated from Crotalus durissus cascavella venom produced renal and vascular effects. NP2_Casca reduced total and proximal sodium tubular transport, leading to an increase in sodium excretion, thereby demonstrating a diuretic action. A hypotensive effect was displayed in an arterial pressure assay, with an increase in nitrite production, suggesting a possible vasoactive action. (C) 2008 Elsevier Ltd. All rights reserved.

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima

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Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom

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Purification, characterization, crystallization and theoretical molecular modeling of gyroxin fraction from Crotalus durissus terrificus venom (Laurenti, 1768)

The venom of Crotalus durissus terrificus snakes presents various substances, including a serine protease with thrombin-like activity, called gyroxin, that clots plasmatic fibrinogen and promote the fibrin formation. The aim of this study was to purify and structurally characterize the gyroxin enzyme from Crotalus durissus terrificus venom. For isolation and purification, the following methods were employed: gel filtration on Sephadex G75 column and affinity chromatography on benzamidine Sepharose 6B; 12% SDS-PAGE under reducing conditions; N-terminal sequence analysis; cDNA cloning and expression through RT-PCR and crystallization tests. Theoretical molecular modeling was performed using bioinformatics tools based on comparative analysis of other serine proteases deposited in the NCBI (National Center for Biotechnology Information) database. Protein N-terminal sequencing produced a single chain with a molecular mass of similar to 30 kDa while its full-length cDNA had 714 bp which encoded a mature protein containing 238 amino acids. Crystals were obtained from the solutions 2 and 5 of the Crystal Screen Kit (R), two and one respectively, that reveal the protein constitution of the sample. For multiple sequence alignments of gyroxin-like B2.1 with six other serine proteases obtained from snake venoms (SVSPs), the preservation of cysteine residues and their main structural elements (alpha-helices, beta-barrel and loops) was indicated. The localization of the catalytic triad in His57, Asp102 and Ser198 as well as S1 and S2 specific activity sites in Thr193 and Gli215 amino acids was pointed. The area of recognition and cleavage of fibrinogen in SVSPs for modeling gyroxin B2.1 sequence was located at Arg60, Arg72, Gln75, Arg81, Arg82, Lis85, Glu86 and Lis87 residues. Theoretical modeling of gyroxin fraction generated a classical structure consisting of two alpha-helices, two beta-barrel structures, five disulfide bridges and loops in positions 37, 60, 70, 99, 148, 174 and 218. These results provided information about the functional structure of gyroxin allowing its application in the design of new drugs.

Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

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