State of the Art in the Studies on Crotamine, a Cell Penetrating Peptide from South American Rattlesnake

Table 1

Crotamine main characteristics, properties, and potential biotechnological applications*.

Name	Crotamine
Organism	Crotalus durissus terrificus South American rattlesnake; Common name: Cascavel
Taxon authority	[6]
Geographic range	Brazil, Peru, Bolivia, Paraguay, Uruguay, Argentina*
Classification	Myoneurotoxin, cell-penetrating peptide, antimicrobial peptide, and defensin-like peptide
Molecular weight	4726,63 daltons
Isoelectric point	pI 9.54, highly positive
Length	Polypeptide of 42 amino acids
Primary sequence	KQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG
Isoform	Crotamine-Ile 19 (isoleucine substitution at position 19)*
Chemical formula	C346H530N90O82S8 (isoform 1)*
Solubility	Highly soluble in water and physiological solutions
Stability	Highly stable in solution, relative large pH range, and temperature
Folding	Crotamine is arranged in a 𝛼𝛽1𝛽2 topology stabilized by 3 disulfide bridges: an 𝛼-helix with residues 1–7 and a two-stranded antiparallel 𝛽-sheets with residues 9–13 and 34–38.
Disulfide bonds	C4–C36; C11–C30; C18–C37
Physiological and neurological activity	Hind limb paralysis in mice in final concentration 2.5 mg of toxin/kg body mass; Necrosis of muscle cells
Electrophysiology activity	Mammalian Kv1.1, Kv1.2, and Kv1.3 blocker with IC(50) of 286.53±91.72 nM
In vitro toxicity (normal cells)	Nontoxic (concentration ranged from 0.1 to 10 𝜇M)
Embryotoxicity	Nontoxic (concentration ranged from 0.1 to 10 𝜇M)
Cell penetrating activity (in vitro)	Selective: dividing (actively proliferating cells); Concentration, cell type, and cell-cycle dependent
Intracellular localization	Cytosol, vesicles, nucleus, centrioles, and chromosomes
Mechanism of DNA-crotamine interaction	Only electrostatic: crotamine (+charged)—DNA (−charged) aggregate
Uptake	Within 5 minutes, and permanence in the cells for approximately 24 hours
Mechanism of penetration	Membrane heparan sulfate proteoglycans binding and clathrin-dependent endocytosis
Cell penetrating activity (in vivo)	Selective: dividing (actively proliferating cells), for example stem cells in bone marrow, spleen, liver, lung, and so force.
Localization in brain	Able to cross blood-brain barrier, and localization in brain cells
Molecular carrier	Intracellular delivery of DNA (both circular and linear molecules) in vivo and in vitro. Final complex size dependent delivery.
Antimicrobial activity	Modest against Gram-positive and Gram-negative bacteria, with some exceptions, for example Micrococcus luteus, and with no detectable activity against the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 𝜇g/mL.
Antimycotic (-fungal) activity	Significant activity against yeast Candida spp.
Cancer cells toxicity	Toxic (concentration ranged from 0.1 to 10 𝜇M); Inoffensive for normal cells
Anticancer activity (in vitro)	Strong against melanoma cells in vitro
Anticancer activity (in vivo)	Inhibition and delay of melanoma growth in vivo in mouse model
Mechanism of tumor inhibition	Mitochondrial depolarization; Intracellular calcium release
Immunogenicity	Low
Biotechnological and biomedical applications	Marker of centrioles and cell cycle; marker of actively proliferating normal cells; biomolecules carrier; tool for cancer cells investigation; marker of cancer cells in vitro and in vivo, and as antifungal and anticancer agent. Prototype for new drug design.

∗Several old data about crotamine need to be revised using modern approaches.