Bothrops Alternatus Snake Venom Research Articles

Platelet aggregation inhibitors from Bothrops alternatus snake venom

Snake venoms are a complex mixture of proteins and peptides that can activate/inhibit platelet aggregation. Bothrops alternatus venom include three main families: metalloproteinases (SVMPs), serinoproteinases (SVSPs) and phospholipases A2 (PLA2s), among other minor components. In this work, we used inhibitor cocktails (containing Na2-EDTA, PMSF and/or pBPB) to investigate the effect of these three families and of baltergin (a PIII SVMP) on platelet aggregation by a turbidmetric method using a microplate reader. Cocktails 1 (active SVMPs) and 2 (active PLA2s) significantly reduced aggregation induced by ristocetin and collagen and by collagen and thrombin, respectively. Cocktail 3 (active SVSPs) showed a mild activation of aggregation, indicating the content of thrombin-like enzymes (TLEs) in this venom is low. Cocktail 4 (active minor components) displayed inhibitory effect with all agonists assayed (ristocetin, ADP, collagen and thrombin) but at higher IC50 values. Baltergin exhibited inhibitory effect when the catalytic domain was active for ristocetin-stimulated platelet aggregation and showed a non-enzymatic mechanism of inhibition when collagen was used as agonist. It was not able to disaggregate platelet thrombus. We conclude that B. alternatus venom is a source of natural inhibitors of platelet aggregation due to the action of SVMPs and PLA2s. Other minor components such as C-type lectins likely contribute to the antiplatelet effect. The interest in knowing the action of venom components on platelet function lies both in the understanding of the pathophysiology of snake bite envenomation and in their biotechnological application.

Photobiomodulation therapy on local effects induced by juvenile and adult venoms of Bothrops alternatus

Bothrops snake envenomation is characterized by severe local manifestations such as pain, edema, inflammation, hemorrhage, and myonecrosis. Furthermore, it is described that venom from juvenile and adult snakes may have differences in their composition that can lead to differences in the evolution of the clinical manifestation of the victim. Photobiomodulation (PBM) has been shown to be an effective adjuvant therapy to serum therapy to reduce the local effects induced by bothropic snake venom. This study evaluated the effect of PBM on the local reaction, after Bothrops alternatus snake venom (BaV) injection, in its juvenile (BaJV) and adult (BaAV) stages. Balb/C mice were injected with the juvenile or adult venoms of BaV or saline solution (control group). PBM at a wavelength of 660 nm, 100 mW, 0.33 W/cm2, 40 s, and a 0.028 cm2 beam was applied transcutaneous to a single point with a radiant exposure of 4 J/cm2, 30 min after venom injection. Edema, inflammatory infiltrate, hyperalgesia, and myonecrosis were analyzed. Both venoms induced significant edema and myonecrosis in the gastrocnemius muscle. Hyperalgesia in the mice paw and a prominent leukocyte infiltrate into the peritoneum were also observed. PBM significantly reduced all evaluated parameters. In conclusion, PBM treatment was effective in reducing the local effects induced by B. alternatus venom at different stages of snake development and could be a useful tool as an adjuvant treatment for bothropic envenomation.

Effects of cilostazol, a Phosphodiesterase-3 inhibitor, on kidney function and redox imbalance in acute kidney injury caused by Bothrops alternatus venom

The mechanisms of pathogenesis of acute kidney injury (AKI) in snakebites is multifactorial and involves hemodynamic disturbances, with release of free radical causing cytotoxic effects. The phosphodiesterase-3 (PDE3) inhibitor, Cilostazol, has been reported to provide protection against renal oxidative stress.Objective: We evaluated the protective effects of cilostazol against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity.Methods: Wistar rat kidneys (n = 6, 260–300 g) were isolated and perfused with Krebs-Henseleit solution containing 6 g/100 mL of bovine serum albumin. After 30 min, the kidneys were perfused with BaV to a final concentration of 1 and 3 μg/mL, and subsequently evaluated for perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl–). Oxidative stress and renal histological analyses were performed.Results:BaV caused a reduction in all the evaluated renal parameters (PP, RVR, GFR, UF, %TNa+, and %TCl–). Although only the effects on PP and UF were reversed with cilostazol treatment, the decrease in the malondialdehyde levels, without changes in glutathione levels, further reduced the venom-induced renal tissue changes.Conclusion: Our data suggest that PDE3 is involved in BaV-induced nephrotoxicity, as cilostazol administration significantly ameliorated these effects.

Phosphodiesterase-5 inhibitor sildenafil attenuates kidney injury induced by Bothrops alternatus snake venom

Acute kidney injury pathogenesis in envenoming by snakes is multifactorial and involves immunologic reactions, hemodynamic disturbances, and direct nephrotoxicity. Sildenafil (SFC), a phosphodiesterase 5 inhibitor, has been reported to protect against pathological kidney changes. ObjectiveThis study aimed to investigate the protective effect of sildenafil against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity. MethodsKidneys from Wistar rats (n = 6, weighing 260–300 g) were isolated and divided into four groups: (1) perfused with a modified Krebs-Henseleit solution (MKHS) containing 6 g% of bovine serum albumin; (2) administered 3 μg/mL SFC; (3) perfused with 3 μg/mL BaV; and (4) administered SFC + BaV, both at 3 μg/mL. Subsequently, the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl−, respectively) were evaluated. The cyclic guanosine monophosphate (cGMP) levels were analyzed in the perfusate, and the kidneys were removed to perform oxidative stress and histopathological analyses. ResultsAll renal parameters evaluated were reduced with BaV. In the SFC + BaV group, SFC restored PP to normal values and promoted a significant increase in %TNa+ and %TCl−. cGMP levels were increased in the SFC + BaV group. The oxidative stress biomarkers, malondialdehyde (MDA) and glutathione (GSH), were reduced by BaV. In the SFC + BaV group, a decrease in MDA without an increase in GSH was observed. These findings were confirmed by histological analysis, which showed improvement mainly in tubulis. ConclusionOur data suggest the involvement of phosphodiesterase-5 and cGMP in BaV-induced nephrotoxicity since its effects were attenuated by the administration of SFC.

Baltetin: a new C-type lectin-like isolated from Bothrops alternatus snake venom which act as a platelet aggregation inhibiting

C-type lectin-like proteins found in snake venom, known as snaclecs, have important effects on hemostasis through targeting membrane receptors, coagulation factors and other hemostatic proteins. Here, we present the isolation and functional characterization of a snaclec isolated from Bothrops alternatus venom, designated as Baltetin. We purified the protein in three chromatographic steps (anion-exchange, affinity and reversed-phase chromatography). Baltetin is a dimeric snaclec that is approximately 15 and 25 kDa under reducing and non-reducing conditions, respectively, as estimated by SDS-PAGE. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and Edman degradation sequencing revealed that Baltetin is a heterodimer. The first 40 amino acid residues of the N-terminal region of Baltetin subunits share a high degree of sequence identity with other snaclecs. Baltetin had a specific, dose-dependent inhibitory effect on epinephrine-induced platelet aggregation in human platelet-rich plasma, inhibiting up to 69% of platelet aggregation. Analysis of the infrared spectra suggested that the interaction between Baltetin and platelets can be attributed to the formation of hydrogen bonds between the PO32- groups in the protein and PO2- groups in the platelet membrane. This interaction may lead to membrane lipid peroxidation, which prevents epinephrine from binding to its receptor. The present work suggests that Baltetin, a new C-type lectin-like protein isolated from B. alternatus venom, is the first snaclec to inhibit epinephrine-induced platelet aggregation. This could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Evaluation of pro-inflammatory events induced by Bothrops alternatus snake venom

Inflammation is a major local feature of envenomation by bothropic snakes being characterized by a prominent local edema, pain, and extensive swelling. There are reports demonstrating that whole Bothrops snake venoms and toxins isolated from them are able to activate macrophages functions, such as phagocytosis, production of reactive oxygen, cytokines and eicosanoids, however, little is known about the effects of Bothrops alternatus (B.a.) venom on macrophages. In this work, we evaluated the proinflammatory effects of B.a. venom with in vivo and in vitro experiments using the Raw 264.7 cell line and mouse peritoneal macrophages. We detected that B.a. venom augments cell permeability (2-fold), and cellular extravasation (mainly neutrophils), increase proinflammatory cytokines IL1 (∼300-fold), IL12 (∼200-fold), and TNFα (∼80-fold) liberation and induce the expression of enzymes related to lipid signaling, such as cPLA2α and COX-2. Additionally, using lipidomic techniques we detected that this venom produces a release of arachidonic acid (∼10 nMol/mg. Protein) and other fatty acids (16:0 and 18:1 n-9c).Although much of these findings were described in inflammatory processes induced by other bothropic venoms, here we demonstrate that B.a. venom also stimulates pro-inflammatory pathways involving lipid mediators of cell signaling. In this sense, lipidomics analysis of macrophages stimulated with B.a. venom evidenced that the main free fatty acids are implicated in the inflammatory response, and also demonstrated that this venom, is able to activate lipid metabolism even with a low content of PLA2.

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

BackgroundSnake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom.MethodsThe protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane.ResultsBaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO32− groups, present in BaltDC, form hydrogen bonds with the PO2− groups present in the non-lipid portion of the membrane platelets.ConclusionsBaltDC may be of medical interest since it was able to inhibit platelet aggregation.

Efficient muscle regeneration after highly haemorrhagic Bothrops alternatus venom injection

Bothrops alternatus snake venom is particularly characterized for inducing a prominent haemorrhage and affecting hemostasis as a consequence of 43.1% of metallo-proteinases and less than 10% of PLA2 (almost all non-myotoxic phospholipases) in its venomics. In addition, myonecrosis is the major local effect in viper envenoming which might lead to permanent sequela. Then, the rebuilding of the microvasculature at the local injured site acquires significance since represents one of the pivotal stages for subsequent skeletal muscle regeneration either at morphological or functional aspects. Due to the significance played by vasculature in this process, it is important to study by histology and immunohistochemical techniques, the muscular damage and the sequence of skeletal muscle reconstruction (degree of damage, reconstitution of muscle fibres and capillaries). In this work, we injected intramuscularly 50 or 100 μg per mouse of B. alternatus venom in gastrocnemius muscles. We provided a complete description and characterization of the different stages of myogenesis after mild (50 µg) and severe (100 µg) local injury induced by B. alternatus venom toxins. The regeneration was evaluated 24 h, 3, 7, 14 and 28 days after receiving venom injection. Finally, both doses induced an extended necrosis at the site of injection where, when critical steps in the regenerative process are taking place, an efficient tissue rebuilding is achieved. B. alternatus venom is characterized by the high percentage of exclusively class P-III metalloproteinases, and by the lack of class P-I metalloproteinases in its venom composition. This could explain the effectiveness of muscle regeneration after venom injection despite the severity of the initial phase of envenoming.

Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom

BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI⿿5.3, 15% sugar and a related molecular mass of 66,000Da in its monomeric form, and 123,000Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy.

Abstract B64: Alternagin-C, an α2β1 integrin-binding disintegrin-like protein, induces distinct cell responses in fibroblasts, tumor breast and endothelial cells in vitro

Abstract Reciprocal interactions between tumor, stromal cells and the extracellular matrix (ECM) are considered crucial steps for tumor development and progression. Both tumor and stromal cells secrete metalloproteases (MMPs) that modify the tumor microenvironment by degrading the ECM and releasing growth factors that may induce tumor cell proliferation, migration and invasion. MMPs activation is stimulated by co-activation of integrins, major cell-surface adhesion receptors. Disintegrins are strong integrin inhibitors found in snake venoms and used as prototypes for angiogenesis inhibitors. Alternagin-C (Alt-C), a disintegrin-like, Cys-rich protein isolated from Bothrops alternatus snake venom, has strong affinity for α2β1 integrin. ALT-C inhibited adhesion to collagen I of a variety of tumor cell lines, and in low concentrations, it induces VEGF expression and angiogenesis in an in vivo matrigel model. However, its mechanism of action was not very clear and it seems to depend on the cell type. Here, we show that ALT-C inhibited MMP-9 activity and mRNA expression in human breast cancer (MDA-MB-231) and MMP-2 activity in human micro vascular endothelial cells (HMEC-1) conditioned media from monocultures. ALT-C also increased the expression of angiogenic genes such as VEGF-A in fibroblasts and tumor cells but not in HMEC-1. However, ALT-C upregulated c-MYC mRNA expression only in tumor cells. ALT-C (10, 40, 100 and 1000 nM) also inhibited transendothelial migration of MDA-MB-231 cells through a monolayer of HMEC-1. In conclusion, these results suggest that α2β1 integrin binding by ALT-C may induce distinct effects in different cell lines by modulating MMP activity and stimulating the expression of angiogenic factors that influence tumor progression. These results may be very useful for medical and biotechnological applications in the development of anti-angiogenic and anti-metastatic therapies. Support: FAPESP, CNPq, CAPES (Brazil) Citation Format: Lívia Mara Santosa, Tamires de Castro Vieira, Heloísa Sobreiro Selistre-de-Araújo. Alternagin-C, an α2β1 integrin-binding disintegrin-like protein, induces distinct cell responses in fibroblasts, tumor breast and endothelial cells in vitro. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B64. doi:10.1158/1538-7445.CHTME14-B64

Molecular characterization of metalloproteases from Bothrops alternatus snake venom

We have previously demonstrated that alternagin-C (ALT-C), a disintegrin-like, Cys-rich protein isolated from Bothrops alternatus snake venom, induces human vascular endothelial cell (HUVEC) proliferation and angiogenesis in in vitro and in vivo assays. Therefore this protein could be interesting as a new approach for tissue regeneration studies. However, its primary sequence was not completely determined since the protein isolated from crude venom is usually a mixture of isoforms. Here we describe the transcriptome analysis of B. alternatus from the venom glands of a single male specimen. About 800 good-quality contigs were screened for snake venom metalloproteases/disintegrins, resulting in the following expression profile for these enzymes: 4% for P-I, 7% for P-II and 89% for P-III SVMPs. The PII-SVMP sequence code for RGD-disintegrins and all the expressed PIII-sequences have the ECD adhesive motif. A cDNA sequence coding for an ALT-C homolog was completely sequenced and characterized. Comparative sequence and structural analyses suggested new features that distinguish SVMP classes such as two prolyl endopetidase cleavage sites. All these data add new information on the expression pattern of metalloproteases of B. alternatus venom and may have practical applications for the production of recombinant disintegrins for cell adhesion studies.

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A2 (PLA2), and BaltTX-II, an Asp49 catalytically active PLA2 isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA2 BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA2, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA2s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.

Histological and Ultrastructural Analyses of Muscle Damage Induced by a Myotoxin Isolated from Bothrops alternatus Snake Venom

Muscular necrosis is a serious consequence of Bothrops snake bites that may lead to permanent loss of tissue or function. Myonecrosis may be due to injury to blood vessels, destabilization and/or rupture of plasma membrane, and inflammatory mechanisms triggered by different proteins from the snake venom. In this work we describe the isolation and partial functional characterization of a myotoxin from B. alternatus snake venom. The myotoxin was isolated by a combination of ion exchange and gel filtration chromatography and displayed a molecular weight of approximately 15,000, as estimated by SDS-PAGE under reducing conditions. In non-reducing conditions a protein band of approximately 25,000 was also observed, suggesting that its native form is a homodimer. The myotoxin induced myonecrosis, but had no proteolytic and phospholipase A₂ activities. The myotoxic activity was assessed on the basis of the histological and ultrastructural alterations induced by the toxin in the gastrocnemius skeletal muscle of Swiss mice. The toxin led to a series of drastic degenerative events characterized by extensive cellular destruction, loss of the arrangements of skeletal fibers, intense infiltration of inflammatory cells, fatty degeneration and hemorrhage. Electron microscopy analyses revealed that the myotoxin caused cell swelling, mitochondrial alterations and dilation of the sarcoplasmic reticulum, but did not affect the integrity of the muscle cell membranes. The myonecrosis caused by this toxin was related to the perturbation in the membrane permeability, intracellular alterations and inflammatory reaction.

Histological and Ultrastructural Analyses of Muscle Damage Induced by a Myotoxin Isolated from Bothrops alternatus Snake Venom

Muscular necrosis is a serious consequence of Bothrops snake bites that may lead to permanent loss of tissue or function. Myonecrosis may be due to injury to blood vessels, destabilization and/or rupture of plasma membrane, and inflammatory mechanisms triggered by different proteins from the snake venom. In this work we describe the isolation and partial functional characterization of a myotoxin from B. alternatus snake venom. The myotoxin was isolated by a combination of ion exchange and gel filtration chromatography and displayed a molecular weight of approximately 15,000, as estimated by SDS–PAGE under reducing conditions. In non-reducing conditions a protein band of approximately 25,000 was also observed, suggesting that its native form is a homodimer. The myotoxin induced myonecrosis, but had no proteolytic and phospholipase A2 activities. The myotoxic activity was assessed on the basis of the histological and ultrastructural alterations induced by the toxin in the gastrocnemius skeletal muscle of Swiss mice. The toxin led to a series of drastic degenerative events characterized by extensive cellular destruction, loss of the arrangements of skeletal fibers, intense infiltration of inflammatory cells, fatty degeneration and hemorrhage. Electron microscopy analyses revealed that the myotoxin caused cell swelling, mitochondrial alterations and dilation of the sarcoplasmic reticulum, but did not affect the integrity of the muscle cell membranes. The myonecrosis caused by this toxin was related to the perturbation in the membrane permeability, intracellular alterations and inflammatory reaction.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na+/K+-ATPase

BackgroundAcute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na+/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na+/K+-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. MethodsMale Wistar rats were injected with venom (0.8mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72h and 7days post-venom. The rats were then killed and renal Na+/K+-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. ResultsVenom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na+/K+-ATPase α1 subunit were increased 6h post-venom, whereas Na+/K+-ATPase activity increased 6h and 24h post-venom. ConclusionsBothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na+/K+-ATPase expression and activity in the early phase of renal damage. General significanceEnhanced Na+/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.

Rat skin wound healing induced by alternagin‐C, a disintegrin‐like, Cys‐rich protein from Bothrops alternatus venom

Alternagin-C (ALT-C) is a disintegrin-like, Cys-rich protein isolated from Bothrops alternatus snake venom, which has been shown to induce in vivo angiogenesis. Therefore, this protein could be interesting as a new approach for tissue regeneration studies. Here the effects of ALT-C on fibroblasts and inflammatory cells, collagen type III and type I and TGF-α expression in a rat wounded skin model were studied. Thirty-five male Wistar rats (weight 270 ± 20 g) were divided into seven groups with five animals in each of the following groups: a control group which wounded animals received treatment with natrozol(®) gel only; ALT-C10, ALT-C60 and ALT-C100 groups of wounded animals that were treated with the same amount of gel containing 10, 60 and 100 ng of ALT-C, respectively. Animals were treated once a day with 20 µl of gel associated or not with ALT-C for 1, 3, 5 or 7 days. ALT-C treatment increased the fibroblast density, collagen deposition and accelerated the inflammatory process, mostly in the ALT-C60 group. These results indicate that ALT-C improves wound repair process in rat skin. Thus, ALT-C could be a candidate to the development of a novel therapeutic strategy for wounded skin repair.

Isolation and functional characterization of a new acidic PLA 2 Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina

An acidic protein with phospholipase A 2 activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A 2 in correspondence with Asp49 PLA 2 group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA 2 revealed a high degree of homology sequence (90–56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 μg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 μg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) “de novo amino acid sequencing”, also provides more database about the small group of the non-myotoxic PLA 2s isolated up to the present.

Platelet Aggregation and Antibacterial Effects of an L‐Amino Acid Oxidase Purified from Bothrops alternatus Snake Venom.

Abstract The isolation and biochemical/enzymatic characterization of an l -amino acid oxidase, Balt-LAAO-I, from Bothrops alternatus snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI ∼ 5.37, homodimeric, Mr∼123,000, whose N-terminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Balt-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Balt-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications.

Platelet aggregation and antibacterial effects of an l-amino acid oxidase purified from Bothrops alternatus snake venom

The isolation and biochemical/enzymatic characterization of an l-amino acid oxidase, Balt-LAAO-I, from Bothrops alternatus snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, p I ∼ 5.37, homodimeric, M r∼123,000, whose N-terminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Balt-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Balt-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications.

The Disintegrin-like Domain of the Snake Venom Metalloprotease Alternagin Inhibits α2β1 Integrin-Mediated Cell Adhesion

The α2β1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Here we describe the isolation of a novel metalloproteinase/disintegrin, which is a potent inhibitor of the collagen binding to α2β1 integrin. This 55-kDa protein (alternagin) and its disintegrin domain (alternagin-C) were isolated from Bothrops alternatus snake venom. Amino acid sequencing of alternagin-C revealed the disintegrin structure. Alternagin and alternagin-C inhibit collagen I-mediated adhesion of K562-α2β1-transfected cells. The IC50 was 134 and 100 nM for alternagin and alternagin-C, respectively. Neither protein interfered with the adhesion of cells expressing αIIbβ3, α1β1, α5β1, α4β1 αVβ3, and α9β1 integrins to other ligands such as fibrinogen, fibronectin, and collagen IV. Alternagin and alternagin-C also mediated the adhesion of the K562-α2β1-transfected cells. Our results show that the disintegrin-like domain of alternagin is responsible for its ability to inhibit collagen binding to α2β1 integrin.