Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC from Crotalus durissus terrificus (South American rattlesnake) venom and their action on insulin secretion by pancreatic islets

Crotamine, a neurotoxin present in the venom of the South American rattlesnake Crotalus durrisus terrificus exists as several polymorphic variants, as demonstrated by recombinant DNA technology (Smith and Schmidt, Toxicon 28 (1990) 575-585). We have isolated native crotamine by chromatography on Sephadex G75, and have purified two crotamine isoforms (F2 and F3) by a single step of RP-HPLC. Native crotamine and RP-HPLC fractions F2 and F3 produced skeletal muscle spasms and spastic paralysis in mice. At low glucose concentrations (2.8-5.6 mmol/l), none of the crotamines altered the insulin secretion by rat isolated islets. In the presence of 16.7 mmol glucose/l, F2 (5 microg/ml), but not F3, increased insulin secretion two-fold, whereas native crotamine (1.5, 5 and 16.5 microg/ml) potentiated the secretion dose-dependently. The increase in insulin secretion induced by F2 fraction (5 microg/ml) was similar to that obtained with 16.5 microg of native crotamine/ml. These results indicate that the mode of action of the F2 and F3 isoforms in beta-cells is different from that in muscle cells. This difference may be related to the binding affinity of each isoform for the Na(+) channels located in the beta-cell membrane. Crotamine isoforms may be valuable tools for studying the involvement of Na(+) channels in the mechanism of insulin secretion.     

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.     

Structure-function relationship of myotoxin a using peptide fragments.

Myotoxin a, a small basic polypeptide isolated from the venom of prairie rattlesnake (Crotalus viridis viridis), has been shown to bind to sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The attachment of myotoxin a to Ca(2+)-ATPase is believed to cause uncoupling of the calcium pump. In order to further elucidate which portion of myotoxin a is important for the uncoupling action, five peptides were synthesized and two peptide fragments were obtained by chemical cleavage. These peptides correspond to discrete portions of the primary sequence of myotoxin a. The peptides are equivalent to the primary sequence of myotoxin a from 1 to 16 residues, 7 to 22 residues, 13 to 28 residues, 19 to 34 residues, and 25 to 42 residues. Chemically produced fragments are equivalent to 1 to 28 residues and 29 to 42 residues of myotoxin a. Peptides of the sequences "YKQCHKKGGHCFPKEK" and "LGKMDCRWKWKCCKKGSG" of myotoxin a inhibited 45Ca uptake into isolated SR and bound to Ca(2+)-ATPase. The same peptides caused weak skeletal muscle vacuolization similar to that caused by native myotoxin a and increased serum creatine kinase activity. The active peptides correspond to the N-terminal and C-terminal portions of myotoxin a. The inactive or less active peptides have sequences which correspond to the middle sequence of myotoxin a. From this study, both the N-terminal and the C-terminal regions of primary sequence of myotoxin a are required to express myotoxin a's biological activity.     

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.     

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.     

A new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis)

Fast atom bombardment (FAB) mass spectrometry was used to identify a new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis). FAB mass spectrometry and Edman degradation were used to characterize its structure. This toxin is similar to myotoxin I from C. v. concolor, except that it possesses an additional. C-terminal asparaginyl-alanine. At 45 residues it is the longest known myotoxin a homolog. A myotoxin of 43 residues, identical to myotoxin I from C. v. concolor, was also found. To date no other species has been shown to produce more than one length of myotoxin. The present paper documents 42-, 43-, and 45-residue myotoxins from the venom of a single animal.     

Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus)

1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS-PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. Results indicate that "collilineatus toxin" is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.     

Biochemical characterization of hemorrhagic toxin from crotalus viridis viridis (prairie rattlesnake) venom

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viridis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose column chromatographies.The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyacrylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 dallons, and it contained 471 amino acid residues. The toxin possessed hemorrhagic activity with a minimum hemorrhagic dose (MHD) of 0.11 μ g, and hydrolytic activity on dimethylcasein, casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorrhagic and casein hydrolytic activities were inhibited by EDTA, o-phenanthroline or dithiothreitol. The toxin contained 1 mole of zinc per mole of protein and zinc is essential for both hemorrhagic and proteolytic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin also hydrolyzed Aα and Bβ chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase activity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated from C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.     

Mechanisms controlling venom expulsion in the western diamondback rattlesnake, Crotalus atrox

Although many studies have documented variation in the amount of venom expended during bites of venomous snakes, the mechanistic source of this variation remains uncertain. This study used experimental techniques to examine how two different features of the venom delivery system, the muscle surrounding the venom gland (the Compressor Glandulae in the rattlesnake) and the fang sheath, could influence venom flow in the western diamondback rattlesnake, Crotalus atrox. Differential contraction of the Compressor Glandulae explained only approximately 30% of the variation in venom flow. Lifting (compression) of the fang sheath as occurs during a normal strike produced marked increases in venom flow; these changes were closely correlated and exceed in magnitude by almost 10 x those recorded from the Compressor Glandulae alone. These results suggest that variation in these two aspects of the venom delivery system--both in terms of magnitude and temporal patterning--explain most of the observed variation in venom injection. The lack of functional or mechanical links between the Compressor Glandulae and the fang sheath, and the lack of skeletal or smooth muscle within the fang sheath, make it unlikely that variation in venom flow is under direct neural control. Instead, differential venom injection results from differences in the pressurization by the Compressor Glandulae, the gate keeping effects of the fang sheath and enclosed soft-tissue chambers, and by differences in the pressure returned by peripheral resistance of the target tissue.     

Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins

Cysteine-rich secretory proteins (CRISPs) are found in epididymis and granules of mammals, and they are thought to function in sperm maturation and in the immune system. Recently, we isolated and obtained clones for novel snake venom proteins that are classified as CRISP family proteins. To elucidate the distribution of snake venom CRISP family proteins, we evaluated a wide range of venoms for immuno-cross-reactivity. Then we isolated, characterized, and cloned genes for three novel CRISP family proteins (piscivorin, ophanin, and catrin) from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus), king cobra (Ophiophagus hannah), and western diamondback rattlesnake (Crotalus atrox). Our results show the wide distribution of snake venom CRISP family proteins among Viperidae and Elapidae from different continents, indicating that CRISP family proteins compose a new group of snake venom proteins.     

Evaluation of the genotoxicity of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes

In the present study, experiments were carried out to evaluate the mutagenic potential and genotoxic effects of Crotalus durissus terrificus snake venom and its isolated toxins on human lymphocytes, using the micronucleus and comet assays. Significant damage to DNA was observed for crotoxin and crotapotin (CA). Basic phospholipase A(2) (CB) and crotamine did not present any mutagenic potential when evaluated by the micronucleus test. C. d. terrificus crude venom was able to induce the formation of micronuclei, similarly to the mutagenic drug used as a positive control. In the comet assay, all the toxins tested (crotamine, crotoxin, CB and CA) and C. d. terrificus venom presented genotoxic activity. Studies on the cytogenetic toxicology of animal venoms and their isolated proteins are still very scarce in the literature, which emphasizes the importance of the present work for the identification and characterization of potential therapeutic agents, as well as for the better understanding of the mechanisms of action of toxins on the human body.     

Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus

We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of kappa- and delta-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be 相似文献   

Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox.

Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.     

Vascular effects and electrolyte homeostasis of the natriuretic peptide isolated from Crotalus oreganus abyssus (North American Grand Canyon rattlesnake) venom

Crotalus oreganus abyssus is a rattlesnake that is usually found in the Grand Canyon, United States of America. Knowledge regarding the composition of C. o. abyssus venom is scarce. New natriuretic peptides (NPs) have been isolated and characterized from the venoms of members of the Crotalinae family. The NP family comprises three members, ANP (atrial natriuretic peptide), BNP (b-type natriuretic peptide) and CNP (c-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. The aim of the present study was to characterize a novel natriuretic-like peptide (Coa_NP2), isolated from C. o. abyssus venom. The Coa_NP2 presents an average molecular mass of 3419.88Da (theoretical average molecular mass 3418.94Da, monoisotopic molecular mass 3416.66Da and theoretical PI 7.78) and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides. The peptide has 32 amino acids and its complete sequence is SYGISSGCFGLKLDRIGTMSGLGCWRLLQDSP. Coa_NP2 is a natriuretic peptide of the ANP/BNP-like family, since the carboxyterminal region of CNP has its own NP domain. We demonstrate, herein, that Coa_NP2 produces a dose-dependent decrease in mean arterial pressure in rats, followed by significant increases in concentrations of markers of nitric oxide formation measured in the plasma and vasorelaxation in a thoracic aortic ring bath. The structural and biological aspects confirm Coa_NP2 as a new natriuretic peptide, isolated from snake venom.     

Specificity of hemorrhagic proteinase from Crotalus atrox (western diamondback rattlesnake) venom

Hemorrhagic proteinase, HTb, isolated from Crotalus atrox (western diamondback rattlesnake) venom was studied for its specificity. HTb showed fibrinogenase activity, hydrolyzing the A alpha chain of fibrinogen first, followed by the cleavage of the B beta chain. HTb is different from thrombin and did not produce a fibrin clot. The degradation products of fibrinogen were found to be different, indicating that the cleavage sites in the A alpha and B beta chains are different from those of thrombin. N-Benzoyl-Phe-Val-Arg-p-nitroanilide was not hydrolyzed by HTb, although this substrate was hydrolyzed by thrombin and reptilase.     

Isolation,enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A₂ (PLA₂). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca²⁺ and Mg²⁺) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA₂ through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA₂, thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.     

Isolation and characterization of a hemorrhagic proteinase from timber rattlesnake venom

A protein isolated from timber rattlesnake (Crotalus horridus horridus) venom by ion-exchange and high-pressure liquid chromatography is hemorrhage inducing and lethal to mice (LD50 of 10 micrograms/g of body weight). It is a Ca2+- and Zn2+-containing proteinase and has the ability to hydrolyze hide powder azure. Atomic absorption spectroscopy shows 2.5 Ca2+ and 1 Zn2+ per protein monomer. The proteinase activity is destroyed by incubation with disulfide-reducing agents and by dialysis against ethylenediaminetetraacetate. Coincident with the loss of proteinase activity is a corresponding loss of lethal and hemorrhagic activities, suggesting that all three are related. Attempts to replace the metals and restore activity have been unsuccessful. Amino acid analysis and isoelectric focusing reveal that this component is an acidic protein (pI = 5.1) containing about 20 disulfide bonds and 507 residues. Reduction of one disulfide bond per molecule decreases proteinase activity by 50% while reduction of eight disulfide bonds decreases activity by 80%. Loss of hemorrhagic activity parallels the decrease in proteinase activity.     

Inhibition of malaria parasite Plasmodium falciparum development by crotamine,a cell penetrating peptide from the snake venom

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC₅₀ value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H⁺ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.     

Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.     

Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom.

Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 micro g per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal alpha-helix and a small antiparallel triple-stranded beta-sheet arranged in an alphabeta1beta2beta3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an alpha/beta fold, though with a beta1alphabeta2beta3 topology. In addition, as the antibacterial beta-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human beta-defensins shows a similar fold and a comparable net positive potential surface. To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel.