Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom
Introduction
PLA2 are classified into three groups, based on their primary structure, group I includes mammalian pancreatic and Elapidae snake venom PLA2, group II is represented by the mammalian non-pancreatic and Viperidae snake venom PLA2 and group III consists of bee and lizard venom PLA2 (Dennis, 1994). PLA2 can also be classified as monomeric, homomultimeric and heteromultimeric. The last of these groups includes crotoxin, the principal neurotoxin of Crotalus durissus terrificus (South American rattlesnake) venom. Crotoxin consists of two non-identical subunits, a weakly toxic basic PLA2 subunit and a non-enzymatic subunit (crotapotin). Crotapotin acts as a chaperon protein for PLA2 to increase the neurotoxicity and decrease the catalytic activity of this enzyme (Soares et al., 2001). Thus, crotapotin is a natural inhibitor of crotalic and other PLA2.
In addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxicity, myotoxicity, edema formation, platelet aggregation, cardiotoxicity and anticoagulant action have been attributed to PLA2 (Soares et al., 2001). These enzymes also affect cell signaling, proliferation and migration and have an antimicrobial action (Lambeau and Lazdunski, 1999, Valentin and Lambeau, 2000). We reported that the venom of Crotalus durissus cascavella, a subspecies of C.durissus common in northeastern Brazil, causes renal lesions by a direct action on tubule and glomerular cells (Martins et al., 1998). In the present study, we investigated the effects of crotapotin, obtained from crotoxin isolated from C.d. cascavella venom on renal function in the isolated rat kidney. The antimicrobial, inflammatory and enzyme inhibiting activities of crotapotin were also evaluated.
Section snippets
Venom, chemicals and reagents
C.d. cascavella venom was a gift from the Regional Snake Laboratory of Fortaleza (LAROF), Ceará. PLA2 of Crotalus durissus collilineatus and Crotalus durissus terrificus PLA2 were previously purified on the Protein Chemistry Laboratory (Department of Biochemistry, UNICAMP). All chemicals and reagents used in this work were of analytical or sequencing grade.
Reverse phase HPLC
Twenty milligrams of desiccated venom were dissolved in 750 μl of 0.1% (v/v) trifluoroacetic acid (solvent A). The resulting solution was
Purification and biochemical characterization of crotapotin from C.d. cascavella venom
C.d. cascavella venom contained several Crtp isoforms (F2–F5) (Fig. 1a). Fraction F3 was the main isoform, with ∼60% homology with the other crotapotins in the venom. This fraction was repurified and yielded a single peak (insert, Fig. 1a). Reduced and carboxymethylated F3 showed three major peaks identified as chains A, B and C (Fig. 1b).
The primary structures of chains A, B and C were deduced by N-terminal sequencing following digestion with protease V8 (Fig. 2a). All chains showed a high
Discussion
Crotoxin is composed of two non-covalently associated subunits; a basic and weakly toxic phospholipase A2, subunit B (PLA2) and an acidic protein, subunit A (crotapotin), which is non toxic and devoid of enzymatic activity. However, crotapotin, separately from the crotoxin complex has shown many biological activities (Landucci et al., 2000).
The edematogenic activity induced by PLA2 was not decreased or inhibited by the addition of crotapotin from the C.d. cascavella, which may be associated to
References (29)
- et al.
Myonecrosis, myoglobinuria and acute renal failure induced by South American rattlesnake (Crotalus durissus terrificus) envenomation in Brazil
Toxicon
(1985) - et al.
Antimicrobial peptides from the Brazilian frog Phyllomedusa distincta
Peptides
(1999) - et al.
A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtti: full characterization and interaction with targed organisms
Biochim. Biophys. Acta
(1998) Gluconeogenesis in the isolated perfused rat kidney
J. Biol. Chem.
(1970)- et al.
Chromogenic substrates and assay of phospholipases A2
Meth. Enzymol.
(1991) Diversity of group types, regulation and function of phospholipase A2
J. Biol. Chem.
(1994)- et al.
Simple and inexpensive membrane lung for small organ perfusion
J. Lipid. Res.
(1974) - et al.
Citrate, pyruvate and lactate contaminants of commercial serum albumin
J. Lipid. Res.
(1968) - et al.
Receptor for a growing family of secreted phospholipase A2
Trends. Pharmacol. Sci.
(1999) - et al.
Effect of crotapotin and heparin on the rat paw oedema induced by different secretory phospholipase A2
Toxicon
(2000)