Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum.

Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation

Purification of γBjPLI from <i>B</i>. <i>jararaca</i> serum.

<p>Purification of γBjPLI from <i>B</i>. <i>jararaca</i> serum.</p

Purification of γBjPLI.

<p>A) Fractionation of <i>B</i>. <i>jararaca</i> serum on ion exchange column (DEAE). Elution was initially done keeping buffer A (25 mM Tris, pH 7.5), 10% of buffer B (25 mM Tris, 1M NaCl pH 7.5) and then making a gradient of B up to 50% (500 mM NaCl), with a flow rate of 1 mL/min. B) Elution of the fractions of pool D2 applied to an affinity column (CNBr-activated Sepharose + crotoxin), done with 1 M glycine pH 2 (indicated by an arrow). SDS-PAGE: 1. Molecular mass marker (Dual Color Precision Plus, BioRad) 2. γBjPLI with β-mercaptoethanol; 3. γBjPLI without β-mercaptoethanol.</p

Proteins identified with higher percentage of coverage from the peptides obtained from the band excised from SDS-PAGE and analyzed by mass spectrometry in Synapt G2 HDMS (Waters).

<p>Proteins identified with higher percentage of coverage from the peptides obtained from the band excised from SDS-PAGE and analyzed by mass spectrometry in Synapt G2 HDMS (Waters).</p

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from <i>Bothrops jararaca</i> snake serum

<div><p>Phospholipases A₂ (PLA₂) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from <i>Bothrops jararaca</i> serum, named γBjPLI. PLA₂ inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA₂ was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation.</p></div

Alignment of the primary structure of γBjPLI and A8I4L6_BOTJA.

<p>Alignment of the γPLI amino acid sequence of <i>B</i>. <i>jararaca</i> (A8I4L6_BOTJA) with the peptides acquired by mass spectrometry (γBjPLI). The 16 Cys are in red, the 3 possible phosphorylation sites are indicated in blue and the N-glycosylation site is indicated in green. The oligopeptide ¹⁰⁴QPFPGLPLSRPNGYY¹¹⁸ is surrounded by a rectangle. The symbol (*) indicates conserved residues; (.) indicates semi-conservative mutations. The signal peptide is highlighted in gray.</p

Biological activity of γBjPLI (<i>in vivo</i>).

<p>A) Neutralization of the myonecrotic activity of γBjPLI caused by PLA₂. Samples of saline (20 μL), PLA₂ (20 μg), γBjPLI (20 μg) or PLA₂ + γBjPLI (20 μg + 20 μg) were preincubated for 30 min at 37°C, injected into the <i>gastrocnemius</i> muscle and then the CK present in the mice blood (Swiss) was quantified and expressed in U/L. B) Neutralization of edematogenic activity of PLA₂. Samples of PLA₂ (10 μg), γBjPLI (10 μg) or PLA₂ + γBjPLI (10 μg + 10 μg), previously incubated for 30 min at 37°C, were injected into the right sub plantar region of the mice paw (Swiss). The volume of edema was monitored using a hydroplethysmometer, until the decrease of the inflammation reached 20% of the initial one.</p