Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the Crotalus durissus cumanensis Venom

Abstract

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its "in vitro" neurotoxic, myotoxic and lethality (DL(50)) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (mu-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD(50) of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.

Biochemical Characterization and Pharmacological Properties of New Basic PLA2BrTX-I Isolated fromBothrops roedingeri(Roedinger's Lancehead) Mertens, 1942, Snake Venom

“…The purification procedure for basic PLA 2 s developed by Ponce-Soto et al [6, 15, 23, 24] showed to be also efficient for the obtainment of the Bothropsroedingeritoxin I PLA 2 (BrTX-I) from Bothrops roedingeri snake venom. Fractionation protocol of this crude venom using a single pass chromatographic in a column μ -Bondapack C-18 coupled to a system of reverse phase HPLC (0.78 cm–30 cm; Waters 991-PDA system) gave rise to 15 fractions at 280 nm, the eight last being the basic PLA 2 named BrTX-I (Figure 1).…”

“…The ability of BrTX-I to induce edema was studied in groups of five Swiss mice (18–20 g) according Ponce-Soto et al [6, 23, 24]. Twenty microliters of phosphate-buffered saline (PBS; 0.12 M NaCl, 0.04 M sodium phosphate, pH 7.2) with BrTX-I (1, 5, 10 and 20  μ g/paw) were injected in the subplantar region of the right footpad.…”

“…Snake venom PLA 2 s displays a variety of activities, such as neurotoxicity, myotoxicity, cardiotoxicity, and hemolysis that may be modulated by specific receptors located on target cells [3–6]. Indeed, PLA 2 receptors classified as kinds M and N [7] have been identified in various kinds of cells, including vascular smooth muscle cells, platelets, neutrophils, chondrocytes, fibroblasts, hepatocytes, and mesangial cells, as well as in brain, lung, and skeletal muscle [8, 9].…”

“…Indeed, PLA 2 receptors classified as kinds M and N [7] have been identified in various kinds of cells, including vascular smooth muscle cells, platelets, neutrophils, chondrocytes, fibroblasts, hepatocytes, and mesangial cells, as well as in brain, lung, and skeletal muscle [8, 9]. Snake venom PLA 2 can bind to M receptors, which are the most common kind found in human macrophages and muscle cells, and these may mediate some of the deleterious actions of venom PLA 2 s, although that was not conclusively demonstrated [5, 6]. …”

Renal and vascular effects of Crotalus durissus cumanensis venom and its crotoxin fraction

“…Crotalus durissus cumanensis (CDCm) is a snake found in Venezuela and northern Brazil, and its venom contains enzymes and toxins such as crotoxin, crotamine, gyroxin and convulxin (10,11). Crotoxin, the major component of the C. d. cumanensis venom (CDCmV), is responsible for most of its neurotoxic and myotoxic effects (6,12).…”