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Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

Furtado JL, Oliveira GA, Pontes AS, Setúbal Sda S, Xavier CV, Lacouth-Silva F, Lima BF, Zaqueo KD, Kayano AM, Calderon LA, Stábeli RG, Soares AM, Zuliani JP - Biomed Res Int (2014)

Bottom Line: The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis.After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min.Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunofarmacologia Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ-Rondônia, Rua da Beira, 7671 Br364, Km 3.5, 76812-245 Porto Velho, RO, Brazil.

ABSTRACT
In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

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(a) Chromatography profile on hydrophobic interaction resin (Butyl-Sepharose HP 50 mm × 10 mm). Fractions 1, 2, and 3 obtained from CM-Sepharose chromatography were pooled and solubilized in 20 mM Ambic plus 4 M NaCl (buffer A) and applied on a Butyl-Sepharose HP column previously equilibrated with the same buffer and eluted with 20 mM Ambic (buffer B) using a step gradient of 0, 25, 50, 75, and 100%. Subsequently, a step using Milli-Q water was performed. (b) High performance chromatographic profile. Fraction 6 obtained from a Butyl-Sepharose HP column was solubilized in 0.1% TFA (solvent A) and applied on a C18 column (discovery 25 mm × 46 mm, 5 μ, 300 Å) previously equilibrated with the same buffer and eluted with 0.1% TFA in 99.9% acetonitrile (solvent B) under a gradient of 0–70% and flow of 1 mL/min. Both elutions were monitored with absorbance at 280 nm.
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fig2: (a) Chromatography profile on hydrophobic interaction resin (Butyl-Sepharose HP 50 mm × 10 mm). Fractions 1, 2, and 3 obtained from CM-Sepharose chromatography were pooled and solubilized in 20 mM Ambic plus 4 M NaCl (buffer A) and applied on a Butyl-Sepharose HP column previously equilibrated with the same buffer and eluted with 20 mM Ambic (buffer B) using a step gradient of 0, 25, 50, 75, and 100%. Subsequently, a step using Milli-Q water was performed. (b) High performance chromatographic profile. Fraction 6 obtained from a Butyl-Sepharose HP column was solubilized in 0.1% TFA (solvent A) and applied on a C18 column (discovery 25 mm × 46 mm, 5 μ, 300 Å) previously equilibrated with the same buffer and eluted with 0.1% TFA in 99.9% acetonitrile (solvent B) under a gradient of 0–70% and flow of 1 mL/min. Both elutions were monitored with absorbance at 280 nm.

Mentions: Bothrops atrox crude venom (100 mg) was applied on a CM-Sepharose ion-exchange column, previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.4, and then eluted with a continuous gradient up to a concentration of 1.0 M of NaCl. Fraction CM-11, with myotoxic activity, was named BaTX-I (Figures 1(a) and 1(c)). The fractions CM-6 and CM-1, with phospholipase A2 activity, were further fractionated on reverse phase C18 (Figure 1(b)) and Butyl-Sepharose (Figure 2(a)) columns, respectively. The fraction CM-6-3 (Figure 1(b)), with PLA2 and myotoxic activities, was named BaTX-II (Figure 1(d)). Fraction CM-1-6 was applied on a RP-HPLC C18 column and resolved into one main peak with only PLA2 activity, named BaPLA2 (Figures 2(b) and 2(c)). The homogeneity of these proteins was further demonstrated by SDS-PAGE. The purified myotoxins consisted of a single polypeptide chain with an apparent approximate molecular weight of 14,500 Da.


Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

Furtado JL, Oliveira GA, Pontes AS, Setúbal Sda S, Xavier CV, Lacouth-Silva F, Lima BF, Zaqueo KD, Kayano AM, Calderon LA, Stábeli RG, Soares AM, Zuliani JP - Biomed Res Int (2014)

(a) Chromatography profile on hydrophobic interaction resin (Butyl-Sepharose HP 50 mm × 10 mm). Fractions 1, 2, and 3 obtained from CM-Sepharose chromatography were pooled and solubilized in 20 mM Ambic plus 4 M NaCl (buffer A) and applied on a Butyl-Sepharose HP column previously equilibrated with the same buffer and eluted with 20 mM Ambic (buffer B) using a step gradient of 0, 25, 50, 75, and 100%. Subsequently, a step using Milli-Q water was performed. (b) High performance chromatographic profile. Fraction 6 obtained from a Butyl-Sepharose HP column was solubilized in 0.1% TFA (solvent A) and applied on a C18 column (discovery 25 mm × 46 mm, 5 μ, 300 Å) previously equilibrated with the same buffer and eluted with 0.1% TFA in 99.9% acetonitrile (solvent B) under a gradient of 0–70% and flow of 1 mL/min. Both elutions were monitored with absorbance at 280 nm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig2: (a) Chromatography profile on hydrophobic interaction resin (Butyl-Sepharose HP 50 mm × 10 mm). Fractions 1, 2, and 3 obtained from CM-Sepharose chromatography were pooled and solubilized in 20 mM Ambic plus 4 M NaCl (buffer A) and applied on a Butyl-Sepharose HP column previously equilibrated with the same buffer and eluted with 20 mM Ambic (buffer B) using a step gradient of 0, 25, 50, 75, and 100%. Subsequently, a step using Milli-Q water was performed. (b) High performance chromatographic profile. Fraction 6 obtained from a Butyl-Sepharose HP column was solubilized in 0.1% TFA (solvent A) and applied on a C18 column (discovery 25 mm × 46 mm, 5 μ, 300 Å) previously equilibrated with the same buffer and eluted with 0.1% TFA in 99.9% acetonitrile (solvent B) under a gradient of 0–70% and flow of 1 mL/min. Both elutions were monitored with absorbance at 280 nm.
Mentions: Bothrops atrox crude venom (100 mg) was applied on a CM-Sepharose ion-exchange column, previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.4, and then eluted with a continuous gradient up to a concentration of 1.0 M of NaCl. Fraction CM-11, with myotoxic activity, was named BaTX-I (Figures 1(a) and 1(c)). The fractions CM-6 and CM-1, with phospholipase A2 activity, were further fractionated on reverse phase C18 (Figure 1(b)) and Butyl-Sepharose (Figure 2(a)) columns, respectively. The fraction CM-6-3 (Figure 1(b)), with PLA2 and myotoxic activities, was named BaTX-II (Figure 1(d)). Fraction CM-1-6 was applied on a RP-HPLC C18 column and resolved into one main peak with only PLA2 activity, named BaPLA2 (Figures 2(b) and 2(c)). The homogeneity of these proteins was further demonstrated by SDS-PAGE. The purified myotoxins consisted of a single polypeptide chain with an apparent approximate molecular weight of 14,500 Da.

Bottom Line: The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis.After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min.Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunofarmacologia Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ-Rondônia, Rua da Beira, 7671 Br364, Km 3.5, 76812-245 Porto Velho, RO, Brazil.

ABSTRACT
In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

Show MeSH
Related in: MedlinePlus