Elsevier

Toxicon

Volume 42, Issue 1, July 2003, Pages 53-62
Toxicon

Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom

https://doi.org/10.1016/S0041-0101(03)00100-4Get rights and content

Abstract

Envenoming by Crotalus durissus subspecies leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure. The most serious systemic alteration and primary cause of death after snakebite is acute renal failure. In this work, we isolated crotapotin, an acid component (Crtp) of crotoxin from Crotalus durissus cascavella venom and we investigated its bactericidal and pro-inflammatory activities as well as its renal effects in rat isolated perfused kidneys. Crtp was bactericidal to the Gram-negative species Xanthomonas axonopodis pv. passiflorae, but was less effective against the Gram-positive Claribacteri ssp, probably because of differences in the cell wall composition. Crtp showed a high amino acid sequence homology with other Crtps described in the literature (around of 90%) and its A and B chains had high conserved regions corresponding to the calcium-binding loop, catalytic site and helix 3 of PLA2. The Crtp showed moderate pro-inflammatory activity and increased significantly the inflammation evoked by PLA2 when co-injected or co-incubated with PLA2. The renal parameters evaluated included the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR) and percent of sodium tubular transport (%TNa+). Crotapotin (5 μg/ml) significantly increased the PP and RVR, whereas the GFR, UF and %TNa+ were unaffected. These results suggest that crotoxin is the main venom component responsible for nephrotoxicity and crotapotin contributes little to this phenomenom. The biological and bactericidal actions of Crtp also suggest that this protein may have functions other than simply acting as a chaperone for PLA2.

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

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