Crotamine Mediates Gene Delivery into Cells through the Binding to Heparan Sulfate Proteoglycans

Abstract

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.

Crotamine, a cell-penetrating peptide, is able to translocate parthenogenetic and in vitro fertilized bovine embryos but does not improve exogenous DNA expression

“…Therefore, the low expression rates achieved after intracytoplasmic injection of crotamine-DNA complex might be explained by the presence of plasmid not completely complexed with the peptide. These results are in contrast with a previous report in mouse embryonic stem cells USP 1 and human carcinoma cells HCT116, in which expression levels reached 20-30 and 98 %, respectively, after transfection with crotamine-plasmid complex of these in vitro cultured cells [9].…”

“…Crotamine-DNA complex formation and stability assay Crotamine-DNA complex was performed as previously described [9]. Crotamine-plasmid DNA (CrP) solution was formed by mixing 2.5 μg of crotamine and 1.0 μg of plasmid in 150 mM NaCl buffer for 15 min.…”

“…For these reasons, it has been successfully used for cell transfection both in vitro and in vivo [9]. These characteristics turn crotamine into an interesting candidate for foreign DNA delivery into preimplantation embryos.…”

Design of a Disulfide-less, Pharmacologically Inert, and Chemically Competent Analog of Maurocalcine for the Efficient Transport of Impermeant Compounds into Cells

“…Recently, a new toxin, crotamin, has been purified from the venom of a South American snake. It also behaves as a CPP but apparently with a preference for dividing cells (8). The great diversity in CPP sequences observed so far suggests that designing new MCa CPP analogues should be easy.…”

Membrane-translocating peptides and toxins: from nature to bedside

“…Moreover, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol. 67 The endosomolytic pathway, used by crotamine to gain access to the cell cytoplasm is another advantage of this natural peptide, that has been explored by several research groups for the effective delivery of drugs. For instance, a penetrating analog named EB1, is protonated in the early-late endosome, forming an amphipathic alpha helix that permeabilize the endosomal membrane.…”