Biochemical characterization and pharmacological properties of new basic PLA2 BrTX-I isolated from Bothrops roedingeri (Roedinger's Lancehead) Mertens, 1942, snake venom

Abstract

BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35-45°C, and required Ca(2+). In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNF α were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.

Figure 1

Elution profile of Bothrops roedingeri venom by RP-HPLC on an m-Bondapack C18 column. Fraction 4 (BrTX-I) contained PLA₂ activity. Insert Re-chromatography on RP-HPLC chromatography of the BrTX-I and electrophoretic profile of BrTX-I with molecular mass ~14 kDa).

Figure 2

Mass determinations of BrTX-I by mass spectrometry, using a Q-Tof Ultima API ESI/MS (TOF MS mode). Insert mass spectrum, showing multiple alkylation channels of alkylated BrTX-I PLA₂ isolated from Bothrops roedingeri.

Figure 3

MS/MS spectrum of the peptide tryptic ion of m/z 1120.310. Ion of the major sequence-specific peptide of the complementing ions YGCYCGWGGR, from which the sequence of BrTX-I tag was deduced.

Figure 4

Alignment of the deduced amino acid sequence of the new PLA₂ BrTX-I with PLA₂ present in venom of PLA₂ (BmTX-I) from Bothrops moojeni [26], PLA₂ PhTX-I from Porthidium hyoprora [27], PLA₂ isoforms (6-1 and 6-2) of the fraction BthTX-II from Bothrops jararacuçu [15], and BbTX-III from Bothrops brazili [27].

Figure 5

(a) PLA₂ activity of Bothrops roedingeri venom and peak 4 (BrTX-I); (b) effect of substrate concentration on the kinetics of BrTX-I (PLA₂) activity. (c) effect of temperature on the PLA₂ activity of BrTX-I; (d) effect of pH on BrTX-I activity; (e) influence of ions (10 mM each) on PLA₂ activity in the absence or presence of 1 mM Ca²⁺. The results of all experiments are the mean ± SE, of three determinations (P < 0.05) and (f) amino acid composition of BrTX-I from Bothrops roendingeri snake venom.

Figure 6

(a) Neuromuscular blockade in chick biventer cervicis muscle preparation (BCP), after addition of B. roedingeri whole venom (50 μg/mL), or fraction BII-4 (BrTX-I; 5, 20, 50, and 100 μg/mL). (b) Inhibition of the response to ACh and KCl, after a 120 min incubation with PLA₂ BrTX-I of Bothrops roedingeri (5, 20, 50, and 100 μg/mL) in chick biventer cervicis muscle preparation. Each point represents the average from five experiments ± SEM. P < 0.05 compared with control. In (c), a group of five Swiss mice (18–20 g) received an intramuscular (i.m.) injection of BrTX-I (1 to 20 μg in 50 μL of PBS), in the gastrocnemius muscle of mice. (d) CK serum levels after control (□) or PLA₂ BrTX-I injection by the i.m. route (○) and i.v route (Δ). At different times, blood was collected, and serum CK levels were measure. Values are means ± SEM of five mice at each point.

Figure 7

In (a), time-course of the mice paw oedema induced by selected doses of BrTX-I (1–20 μg). The oedema, which was expressed as the percentage increased in the volume of the treated group to that of the control group at each time interval, was maximal around 2 h and decreased thereafter. Levels of TNF-α, IL-1 and IL-6 ((b), (c), and (d), resp.) in the serum after injection of BrTX-I. Animals were injected i.m. with BrTX-I (1.0 mg/kg) or sterile saline alone (control) in a final volume of 1 mL. TNF-α, IL-1 and IL-6 ((b), (c), and (d), resp.) were quantified by specific ELISA, in serum collected at the indicated time intervals after BrTX-I or saline injection as described in Section 2. Each bar represents mean GSEM of 5 animals. *P < 0.05 when compared with the corresponding control.