myotoxin-a and crotamine

Toxins have been shown to have many biological functions and to constitute a rich source of drugs and biotechnological tools. We focus on toxins that not only have a specific activity, but also contain residues responsible for transmembrane penetration, which can be considered bioportides-a class of cell-penetrating peptides that are also intrinsically bioactive. Bioportides are potential tools in pharmacology and biotechnology as they help deliver substances and nanoparticles to intracellular targets. Bioportides characterized so far are peptides derived from human proteins, such as cytochrome c (CYCS), calcitonin receptor (camptide), and endothelial nitric oxide synthase (nosangiotide). However, toxins are usually disregarded as potential bioportides. In this review, we discuss the inclusion of some toxins and molecules derived thereof as a new class of bioportides based on structure activity relationship, minimization, and biological activity studies. The comparative analysis of the amino acid residue composition of toxin-derived bioportides and their short molecular variants is an innovative analytical strategy which allows us to understand natural toxin multifunctionality in vivo and plan novel pharmacological and biotechnological products. Furthermore, we discuss how many bioportide toxins have a rigid structure with amphiphilic properties important for both cell penetration and bioactivity.

Topics: Amino Acid Sequence; Animals; Cell-Penetrating Peptides; Crotalid Venoms; Crotalus; Cytochromes c; Drug Delivery Systems; Humans; Models, Molecular; Scorpion Venoms; Scorpions; Toxins, Biological; Viper Venoms; Viperidae

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous β-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

Topics: Animals; Base Sequence; Crotalid Venoms; Crotalus; DNA Copy Number Variations; Genomics; Humans; Mammals; Neurotoxins; Snake Venoms; Transcriptome

Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca

Topics: Amino Acid Sequence; Animals; Cell Line; CHO Cells; Cricetulus; Crotalid Venoms; Crotalus; Mice; Motor Endplate; Muscle, Striated; Peptides

The binding of radiolabeled myotoxin a to various cultured cell lines was evaluated. One rat skeletal muscle-derived cell line, L8, bound substantially more myotoxin a than did all all other cell lines examined. Several biophysical parameters of myotoxin a-L8 binding were determined. Binding was saturable with a moderate binding affinity. Scatchard analysis and Hill plots indicated a single class of binding sites. The binding was reversible, as demonstrated by chase experiments. Radiolabeled myotoxin a bound to the cell surface at a site inaccessible to the general protease, pronase. Specificity and biological relevance of the binding was suggested by competition with unlabeled toxin and various peptides derived from the toxin. Biologically active peptides, corresponding to the N- and C-terminal sequence of myotoxin a, competed with radiolabeled toxin for L8 binding. It was concluded that the L8 system is a suitable cell model to study myotoxin a mechanism of action.

Topics: Amino Acid Sequence; Amino Acids; Animals; Cell Line; Crotalid Venoms; Kinetics; Mice; Molecular Sequence Data; Muscles; Peptides; Pronase; Rats; Thermodynamics

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. The pH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidic pH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for Ile-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.

Topics: Amino Acid Sequence; Crotalid Venoms; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Protein Conformation; Solutions

The potentiating effect of sodium acetate on the toxicity of crotamine from Crotalus durissus terrificus venom, E toxin from Crotalus horridus horridus venom, and myotoxin a from Crotalus viridus viridis venom was examined. Subcutaneous injection of 6.3 mg/kg body weight of either crotamine or E toxin in 0.6 ml of water or myotoxin a in 0.6 ml of 0.05 M Tris/0.1 M NaCl buffer, pH 9.0, failed to produce lethality in mice. Injection of either E toxin or crotamine at doses of 4.0 mg/kg in 0.6 ml of 20 mM phosphate, pH 7.2, containing 1 M sodium chloride also failed to produce lethality. However, when any of the toxins were injected in 0.4 ml of 1 M sodium acetate, pH 7.0, lethality was observed. LD50 values of 1.43 mg/kg for E toxin, 1.39 mg/kg for crotamine and 0.56 mg/kg for myotoxin a were determined under these conditions. Lethality was also observed when either sodium propionate or sodium butyrate was used as a carrier for E toxin. The effect of these two buffers on crotamine and myotoxin a was not examined. Injection of E toxin s.c. in water followed at various time intervals with i.p. injections of 1 M sodium acetate produced lethality, even when the acetate was injected up to 4 hr after the toxin challenge.

Topics: Acetates; Acetic Acid; Animals; Blood; Crotalid Venoms; Drug Synergism; Hydrogen-Ion Concentration; Mice; Mice, Inbred C3H; Pharmaceutical Vehicles

Myotoxin a reduced the resting membrane potential of mouse and rat diaphragms from about -80 mV to -60 mV, induced spontaneous repetitive firing and enhanced the contractile force in response to single stimulations. The depolarizing effect was reversed noncompetitively by tetrodotoxin, local anesthetics or low Na+ solution, but was augmented by ouabain or low Cl-solution while being unaffected by high K+ solution or electrical stimulation of the muscle. The duration of muscle action potential was prolonged by only 20-30%, whereas the rate of rise (dV/dt) was unaffected. About a 40% increase of membrane conductance was observed, be abolished by the Na+-channel blocker tetrodotoxin. By contrast, K+ conductance was unaffected. Effects on caffeine-induced contracture, quantal release of neurotransmitter and the amplitude of miniature endplate potential were not appreciably affected. These effects of myotoxin a indicate that the toxin affects the muscle, but not the nerve, by acting specifically on the Na+-channel of the sarcolemma or T-tubule, like crotamine, rather than on the sarcoplasmic reticulum. The effects of sea anemone toxin II on the Na+-channel (marked depolarization and prolongation of action potential) could not be prevented by saturating the muscle with myotoxin a. On the other hand, the effect of veratridine, a member of another group of toxins acting on the Na+-channel, was enhanced. These results suggest that myotoxin a acts on the Na+-channel at a site which is discrete from those of tetrodotoxin, veratridine and sea anemone toxin II.

Topics: Action Potentials; Animals; Cell Membrane; Cnidarian Venoms; Crotalid Venoms; Diaphragm; Electrophysiology; In Vitro Techniques; Ion Channels; Mice; Mice, Inbred ICR; Muscle Contraction; Muscles; Rats; Sarcolemma; Veratridine