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Partial purification and functional characterization of Ts19 Frag-I, a novel toxin from Tityus serrulatus scorpion venom.

Lima PC, Bordon KC, Pucca MB, Cerni FA, Zoccal KF, Faccioli LH, Arantes EC - J Venom Anim Toxins Incl Trop Dis (2015)

Bottom Line: At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6).We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I.Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP Brazil.

ABSTRACT

Background: The yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.

Methods: The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages.

Results: The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).

Conclusions: We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

No MeSH data available.


Related in: MedlinePlus

Effects of Ts4, Ts3-KS and peak 9.3# on macrophage viability and the cytokine and NO production. Adherent cells were stimulated with Ts4, Ts3-KS and peak 9.3 (50 μg/mL) for 24 h in 5 % CO2 at 37 °C. The supernatants were collected after 24 h. (a) Cell viability was measured by MTT assay. Each column represents the mean ± SEM (n = 6), and the data are from two independent set of experiments (*p < 0.05 compared to control, non-stimulated cells). The concentrations of the cytokines (b) IL-6 and (c) IL-1β in the supernatants were determined by ELISA. The amount of (d) NO2− present in the supernatant was determined by the Griess method. Values are expressed as mean ± SD (n = 4). *p < 0.05 compared to control, non-stimulated cells (ANOVA and Dunnett's post-test). #Peak 9.3: Ts19 Frag-I contaminated with Ts2 and Ts3-KS
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Fig4: Effects of Ts4, Ts3-KS and peak 9.3# on macrophage viability and the cytokine and NO production. Adherent cells were stimulated with Ts4, Ts3-KS and peak 9.3 (50 μg/mL) for 24 h in 5 % CO2 at 37 °C. The supernatants were collected after 24 h. (a) Cell viability was measured by MTT assay. Each column represents the mean ± SEM (n = 6), and the data are from two independent set of experiments (*p < 0.05 compared to control, non-stimulated cells). The concentrations of the cytokines (b) IL-6 and (c) IL-1β in the supernatants were determined by ELISA. The amount of (d) NO2− present in the supernatant was determined by the Griess method. Values are expressed as mean ± SD (n = 4). *p < 0.05 compared to control, non-stimulated cells (ANOVA and Dunnett's post-test). #Peak 9.3: Ts19 Frag-I contaminated with Ts2 and Ts3-KS

Mentions: The toxicity of the toxins Ts3-KS (peak 7.4), Ts19 Frag-I (peak 9.3) and Ts4 (peak 11) at 50 μg/mL was analyzed by MTT assay. We demonstrated that these toxins did not affect J774.1 cell viability when compared to non-stimulated cells (Fig. 4 – a).Fig. 4


Partial purification and functional characterization of Ts19 Frag-I, a novel toxin from Tityus serrulatus scorpion venom.

Lima PC, Bordon KC, Pucca MB, Cerni FA, Zoccal KF, Faccioli LH, Arantes EC - J Venom Anim Toxins Incl Trop Dis (2015)

Effects of Ts4, Ts3-KS and peak 9.3# on macrophage viability and the cytokine and NO production. Adherent cells were stimulated with Ts4, Ts3-KS and peak 9.3 (50 μg/mL) for 24 h in 5 % CO2 at 37 °C. The supernatants were collected after 24 h. (a) Cell viability was measured by MTT assay. Each column represents the mean ± SEM (n = 6), and the data are from two independent set of experiments (*p < 0.05 compared to control, non-stimulated cells). The concentrations of the cytokines (b) IL-6 and (c) IL-1β in the supernatants were determined by ELISA. The amount of (d) NO2− present in the supernatant was determined by the Griess method. Values are expressed as mean ± SD (n = 4). *p < 0.05 compared to control, non-stimulated cells (ANOVA and Dunnett's post-test). #Peak 9.3: Ts19 Frag-I contaminated with Ts2 and Ts3-KS
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666072&req=5

Fig4: Effects of Ts4, Ts3-KS and peak 9.3# on macrophage viability and the cytokine and NO production. Adherent cells were stimulated with Ts4, Ts3-KS and peak 9.3 (50 μg/mL) for 24 h in 5 % CO2 at 37 °C. The supernatants were collected after 24 h. (a) Cell viability was measured by MTT assay. Each column represents the mean ± SEM (n = 6), and the data are from two independent set of experiments (*p < 0.05 compared to control, non-stimulated cells). The concentrations of the cytokines (b) IL-6 and (c) IL-1β in the supernatants were determined by ELISA. The amount of (d) NO2− present in the supernatant was determined by the Griess method. Values are expressed as mean ± SD (n = 4). *p < 0.05 compared to control, non-stimulated cells (ANOVA and Dunnett's post-test). #Peak 9.3: Ts19 Frag-I contaminated with Ts2 and Ts3-KS
Mentions: The toxicity of the toxins Ts3-KS (peak 7.4), Ts19 Frag-I (peak 9.3) and Ts4 (peak 11) at 50 μg/mL was analyzed by MTT assay. We demonstrated that these toxins did not affect J774.1 cell viability when compared to non-stimulated cells (Fig. 4 – a).Fig. 4

Bottom Line: At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6).We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I.Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP Brazil.

ABSTRACT

Background: The yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.

Methods: The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages.

Results: The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).

Conclusions: We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

No MeSH data available.


Related in: MedlinePlus