Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake.
ABSTRACT We report the purification and biochemical/pharmacological characterization of two myotoxic PLA(2) (BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2)) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M(r) approximately 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2), had a sequence of 121 amino acid residues. BbTX-II: [amino acid sequence: see text] with pI value 8.73. BbTX-III: [amino acid sequence: see text] with a pI value of 8.46. BbTX-III presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+). In vitro, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA(2) homologue and BbTX-III PLA(2) induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA(2) homologue, occur in the absence of a measurable PLA(2) activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.
- SourceAvailable from: José María Gutiérrez[show abstract] [hide abstract]
ABSTRACT: The patterns of myotoxicity induced in mice by crotoxin, crotoxin B and a Lys49 phospholipase A(2) (PLA(2)) homologue were compared. Lys49 PLA(2)-induced local myotoxicity is reflected by creatine kinase (CK) loss in injected gastrocnemius muscle, and by a profile of CK increase in plasma characterized by a rapid increment and drop after intramuscular injection, and by a lack of CK increase in plasma after intravenous injection. In contrast, crotoxin and crotoxin B, which induce local and systemic myotoxicity, provoked a more prolonged increment in plasma CK activity upon intramuscular injection, and induced increments in plasma CK after intravenous injection. The three toxins promoted a similar extent of local myotoxicity, assessed by the loss of CK in injected gastrocnemius. A method for the quantitative assessment of the ability of toxins to induce systemic myotoxicity is proposed, based on the estimation of the ratio between the area under the curve in the plasma CK activity (total myotoxicity) to the loss of CK in injected gastrocnemius (local myotoxicity). The highest ratio corresponded to crotoxin, and the lowest corresponded to Lys49 PLA(2), the former being a systemic myotoxin and the latter a local myotoxin. Neutralization by antivenoms also differed between the toxins: a drastic reduction in plasma CK, with very poor neutralization of local CK loss, was achieved in the case of crotoxin B when antivenom was injected intravenously, whereas no neutralization was achieved in the case of Lys49 PLA(2). When tested in undifferentiated myoblasts in culture, Lys49 PLA(2) induced cytotoxicity, whereas crotoxin and crotoxin B did not, evidencing that the latter are devoid of widespread cytolytic activity. Molecular modeling analysis showed that Lys49 PLA(2) has a conspicuous cationic face, which is likely to interact with diverse membranes. In contrast, crotoxin B, despite its overall basic pI, has a lower density of positively charged residues at this molecular region. It is suggested that Lys49 PLA(2)s homologues interact, through this cationic face, with many different cell types, thus lacking specificity for muscle cells. In contrast, crotoxin B has a more selective interaction with targets in the muscle cell membrane. This selectivity might be the basis for the ability of crotoxin and crotoxin B to induce systemic myotoxicity.Toxicon 02/2008; 51(1):80-92. · 2.92 Impact Factor
Article: Lysine 49 phospholipase A 2 proteins[show abstract] [hide abstract]
ABSTRACT: The structures of several K49 PLA2 proteins have been determined and these differ as a group in several regions from the closely related D49 PLA2 enzymes. One outstanding difference is the presence of a high number of positively charged residues in the C-terminal region which combined with the overall high number of conserved lysine residues gives the molecule an interfacial adsorption surface which is highly positively charged compared to the opposite surface of the molecule. Although some nucleotide sequences have been reported, progress in obtaining active recombinant proteins has been slow. The K49 proteins exert several toxic activities, including myotoxicity, anticoagulation and edema formation. The most studied of these activities is myotoxicity. The myotoxicity induced by the K49 PLA2 proteins is histologically similar to that caused by the D49 PLA2 myotoxins, with some muscle fiber types possibly more sensitive than others. Whereas it is clear that the K49 PLA2 myotoxins lyse the plasma membrane of the affected muscle cell in vivo, the exact mechanism of this lysis is not known. Also, it is not known whether the toxin is internalized before, during or after the initial lysis or ever. The K49 PLA2 toxins lyse liposomes and cells in culture and in the latter, the PLA2 myotoxins exert at least two distinct mechanisms of action, neither of which is well-characterized. While the K49 PLA2 proteins are enzymatically inactive on artificial substrates, the toxins cause fatty acid production in cell cultures. Whether the fatty acid release is due to the enzymatic activity of the K49 PLA2 or stimulation of tissue lipases, is unknown. While there may be a role for fatty acid production in one mechanism of myotoxicity, a second mechanism appears to be independent of enzymatic activity. Although we are beginning to understand more about the structure of these toxins, we still know little about the precise mechanism by which they interact with the skeletal muscle cell in vivo.Toxicon 01/1999; 37(3):411-445. · 2.92 Impact Factor
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ABSTRACT: The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-I is a Lys49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (M(r) = 13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2 activity, BthTX-I reveals a high degree of sequence homology with Asp49-PLA2s and with other Lys49-myotoxins. Critical mutations--such as Leu5 for Phe5; Gln11 for X11; Asn28 for Tyr28; Leu32 for Gly32; Lys49 for Asp49; and Asp71 for Asn71--which are apparently involved with the decreasing or elimination of PLA2 activity, have been detected. The same mutations occurred in myotoxin II from Bothrops asper venom, but five extra changes--namely, Pro90 for Ser90; Gly111 for Asn111; His120 for Tyr120; Phe124 for Leu124; and Pro132 for Ala132--have been found relative to myotoxin II.Journal of Protein Chemistry 03/1993; 12(1):57-64.