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

Matias, Mariana Santos; Sousa, Bruna Barbosa de; Pereira, Déborah Fernanda da Cunha; Dias, Edigar Henrique Vaz; Mamede, Carla Cristine Neves; Queiroz, Mayara Ribeiro de; Silva, Anielle Christine Almeida; Dantas, Noélio O.; Soares, Andreimar M.; Costa, Júnia de Oliveira; Oliveira, Fábio de

doi:10.1186/s40409-017-0126-7

Cited by 10 publications

(9 citation statements)

References 45 publications

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“…It is possible that the DC protein is a specific cleavage product of the precursor P-III SVMP from another venom protease that is active in the venom gland, but convincing confirmation of such a mechanism is still not available. On the contrary, for snake venom DC proteins, such as VaaMPIII-3 [10], leberagin-C [11], BaltDC [18], catrocollastatin-C [17], and jararhagin-C [19], the precursor P-III SVMP molecules or the corresponding MP fragments have not been isolated from the venom.…”

Section: Discussionmentioning

confidence: 99%

“…Jararhagin-C (isolated from the venom of Bothrops jararaca) and acucetin (released by autolysis of AaHIV) also affect this process, but their IC 50 values were not determined [12,27]. However, not all DC proteins have this kind of inhibitory action [11,13,14,17,18]. ADP is an important aggregation agonist that triggers platelet activation by binding to P2Y receptors, which leads to secretion of α-granules and exposure of the fibrinogen-binding receptor, integrin α IIb β 3 (GPIIb/GPIIIa), on the surface of platelets.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to VaaMPIII-3, several other DC proteins have been isolated from viperid snake venoms [11][12][13][14][15][16][17][18]. These have shown various pathophysiological effects ex vivo, such as inhibition of platelet aggregation induced by ADP, collagen, and arachidonic acid, inhibition of platelet agglutination induced by ristocetin, chemotactic recruitment of neutrophils, and stimulation of leukocyte rolling in the microcirculation.…”

Section: Introductionmentioning

confidence: 99%

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Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein

Požek

Leonardi

Pungerčar

et al. 2022

Toxins

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Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein

Požek

Leonardi

Pungerčar

et al. 2022

Toxins

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“…Reports indicate that B. alternatus venom damages the endothelium and basement membrane due to the proteolytic action generated by SVMPs (Gay et al, 2009;Palacio et al, 2012;Matias et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Full title: Inhibition of harmful effects from Bothrops alternatus venom by Casearia arborea extract

Chaves¹,

Santos²,

Saturnino³

et al. 2022

JHS

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This study aimed to assess the action of methanol extract from Casearia arborea (Salicaceae) branches against the harmful effects of Bothrops alternatus venom. The venom was tested combined or not with Casearia arborea methanol extract (CAME) at different ratios (1:10, 1:50 (w/w)). Coagulant activity was tested using bovine plasma at 37°C and crude venom (10µg). The hemorrhagic activity of the venom (15µg) was assessed 150 min after inoculating venom samples in the dorsal region of mice. Myotoxic activity was tested by injecting venom into the gastrocnemius muscle of mice, followed by determination of creatine kinase (CK) levels. Phospholipase A 2 activity was evaluated using egg yolk as substrate. Histologic sections of gastrocnemius muscles were analyzed under an optical microscope. The results indicated that CAME inhibited coagulant activity by 59% and reduced hemorrhagic activity by 91%. Myotoxicity analysis demonstrated that serum CK levels declined and phospholipase A 2 activity assessment showed a 50% reduction. The histological sections analyzed indicated decreased local tissue damage in the presence of CAME. These results suggest that CAME may be a potential adjuvant in conjunction with the serum therapy currently applied to neutralize botropic venoms.

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“…As peçonhas são constituídas por uma mistura de proteínas, com atividade enzimática e não enzimática, além de componentes orgânicos (carboidratos, nucleotídeos, aminas, lipídeos e aminoácidos) e inorgânicos (diferentes íons, como cálcio, cobre, ferro, potássio, magnésio, manganês, sódio e zinco) (GOMES, 2013;MATIAS et al, 2017).…”

Section: Lista De Gráficosunclassified

Clonagem e Expressão da Balternina, uma Serinoprotease de Bothrops alternatus (Urutu), em Sistema de Células de Bactéria e Levedura

Polli¹

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Despite their toxicity, snake venoms are composed of a series of organic and non-organic components that show activities against virus and bacteria, as well as therapeutic activities in thrombotic disorders. Thus, the characterization of biomolecules present in venoms has shown to be important to develop alternative therapies. Bhalternin, a toxin from Bothrops alternatus, has been described as a thrombin-like enzyme and has a therapeutic potential in cases of thrombotic disorders. So, the present study had as objective to perform cloning and expression of Bhalternin in bacterial and yeast systems. The mature Bhalternin coding sequence was cloned into pGS-21a e pPICZα A vectors and, after confirmation of the nucleotide sequences by Sanger Method, the plasmids that were constructed, pPGSBH2 e pPαABH9, were utilized in the transformation of Escherichia coli (strain BL21-Codon-Plus(DE3)-RIPL) and the Pichia pastoris (strain X-33), respectively. Different colonies, which showed presence of plasmid DNA by Polymerase Chain Reaction, were purified, submitted to the expression process and, then, evaluated for Bhalternin expression by protein eletrophoresis and Western blotting. In the bacterial system, a protein band corresponding to the expected molecular weight of Bhalternin expressed as fusion protein with glutathione Stransferase (GST) was observed in the supernatant and in the precipitate of bacterial lysate. However, in yeast system, it was not possible to identify a protein band corresponding to the secreted form of Bhalternin. However, in ELISA it was observed levels of expression in yeast system. Still, the coagulation test was carried out and, although a bacterial sample obtained by treating the lysate precipitate with 3 M urea showed activity, the result was inconclusive, since a sample obtained with the control clone (transformed with the vector) also showed activity. In the azocaseinolytic test, all results samples showed activity, including samples obtained with control clones. In the case of yeast expression system, the results seem to indicate low expression of Bhalternin, even though codons optimized for P. pastoris expression were used. On the other hand, in bacterial system, solubilization of Bhalternin with urea was necessary and the treatment may have caused changes in its activities. Therefore, further tests should be performed in order to obtain Bhalternin as soluble form and to evaluate its therapeutic potential in thrombotic disorders, as well as, its antiviral, antibacterial, and antiparasitic activities.

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

Cited by 10 publications

References 45 publications

Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein

Genomic Confirmation of the P-IIIe Subclass of Snake Venom Metalloproteinases and Characterisation of Its First Member, a Disintegrin-Like/Cysteine-Rich Protein

Full title: Inhibition of harmful effects from Bothrops alternatus venom by Casearia arborea extract

Clonagem e Expressão da Balternina, uma Serinoprotease de Bothrops alternatus (Urutu), em Sistema de Células de Bactéria e Levedura

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