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. 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. Cell-penetrating peptides (CPPs) 6The abbreviations used are: CPP, cell-penetrating peptide; HSPG, heparan sulfate proteoglycan; GFP, green fluorescent protein; ES cell, embryonic stem cell; mES cell, mouse ES cell; AP, actively proliferating; BM, bone marrow; GAG, glycosaminoglycan; AO, acridine orange; CHO, Chinese hamster ovary; Cy3-crotamine, Cy3-conjugated crotamine; DIC, differential interference contrast; Fcm, fluorescent confocal microscopy; CD, circular dichroism; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. are molecules that display the distinguishing ability to enter eukaryotic cells through an energy-independent mechanism and to efficiently carry a number of biologically active and therapeutically relevant molecules into the cells. They are represented by multiple sequences of short and positively charged peptides rich in arginine and lysine residues that penetrate through usually impermeable cellular membranes and localize in the cytoplasm or in the nucleus of the cells or both (1Gupta B. Levchenko T.S. Torchilin V.P. Adv. Drug Delivery Rev. 2005; 57: 637-651Crossref PubMed Scopus (575) Google Scholar, 2Zorko M. Langel U. Adv. Drug Delivery Rev. 2005; 57: 529-545Crossref PubMed Scopus (702) Google Scholar, 3Kerkis A. Hayashi M.A.F. Yamane Y. Kerkis I. IUBMB Life. 2006; 58: 1-7Crossref PubMed Scopus (74) Google Scholar). When used as carrier molecules, CPPs are usually covalently linked to the cargo or, alternatively, synthesized or expressed in tandem as a fusion protein with the peptide or protein cargos (4Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 5Rojas M. Donahue J.P. Tan Z. Lin Y.Z. Nat. Biotechnol. 1998; 16: 370-375Crossref PubMed Scopus (142) Google Scholar). A list of cargo molecules delivered into various cells includes peptides, proteins, fragments of DNA, peptide-nucleic acid, small interfering RNA, liposomes, and magnetic nanoparticles (1Gupta B. Levchenko T.S. Torchilin V.P. Adv. Drug Delivery Rev. 2005; 57: 637-651Crossref PubMed Scopus (575) Google Scholar, 2Zorko M. Langel U. Adv. Drug Delivery Rev. 2005; 57: 529-545Crossref PubMed Scopus (702) Google Scholar, 6Schwarze S.R. Dowdy S.F. Trends Pharmacol. Sci. 2000; 21: 45-48Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 7Jarver P. Langel U. Drug Discov. Today. 2004; 9: 395-402Crossref PubMed Scopus (167) Google Scholar). However, particular approaches to attach them to the CPPs are always required. In addition, the usefulness of the CPPs as therapeutic agents has so far been hampered by the unspecific penetration of these carrier molecules into a vast range of cells (8Lindgren M. Hallbrink M. Prochiantz A. Langel U. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 9Lundberg P. Langel U. J. Mol. Recognit. 2003; 16: 227-233Crossref PubMed Scopus (195) Google Scholar). Attempts have been made to confer the required specificity to some CPPs-cargo complexes (10Jiang T. Olson E.S. Nguyen Q.T. Roy M. Jennings P.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 51: 17867-17872Crossref Scopus (720) Google Scholar). The proposed mechanisms by which CPPs penetrate cells as independent molecules or carrying different cargos are still controversial. Several studies have suggested that CPPs translocate into cells via endocytosis (11Richard J.P. Melikov K. Vives B. B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google be A. Trends Biol. 1998; Full Text PDF PubMed Scopus Google Scholar, S. J. 1-7Crossref PubMed Scopus Google Scholar). CPPs with the of the cell followed by to translocate membranes by (11Richard J.P. Melikov K. Vives B. B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). vesicles or heparan sulfate involvement in the uptake mechanisms have also been demonstrated A. P. M. J. 2005; PubMed Scopus Google Scholar). Recently we that crotamine, a from the of South American is a both and non-toxic concentrations crotamine into cells binding to and we have suggested the of crotamine as a marker of centrioles, of cell cycle, and of actively proliferating cells A. Kerkis I. Yamane T. J. 2004; PubMed Scopus Google Scholar). The specificity of crotamine to cells has also been demonstrated by cells The cells with and with fluorescent crotamine followed by with of the and within the nucleus of the cells. Moreover, suggested that crotamine penetration is a cell A. Kerkis I. Yamane T. J. 2004; PubMed Scopus Google Scholar). these that crotamine is from other known natural is a whose is by Z. Chem. PubMed Scopus Google Scholar, Hayashi Yamane T. PubMed Scopus Google Scholar). The of crotamine is by a of A. J. 2003; 270: PubMed Scopus Google Scholar, P. T. Yamane T. K. 2005; PubMed Scopus (74) Google Scholar). The crotamine and other natural (8Lindgren M. Hallbrink M. Prochiantz A. Langel U. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 9Lundberg P. Langel U. J. Mol. Recognit. 2003; 16: 227-233Crossref PubMed Scopus (195) Google Scholar, A. Trends Biol. 1998; Full Text PDF PubMed Scopus Google Scholar, S. J. 1-7Crossref PubMed Scopus Google or CPPs J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Chem. 2000; PubMed Scopus Google to the ability of crotamine to as a delivery In the we crotamine and DNA complexes into a of cells both in vitro and in crotamine uptake and a with cell heparan sulfate proteoglycans followed by endocytosis and peptide accumulation within the vesicles. we demonstrated the of crotamine the of the which in the leakage of these vesicles contents into the cell cytosol. and acridine from and from Crotalus durissus terrificus was from at the and crotamine was as A. Kerkis I. Yamane T. J. 2004; PubMed Scopus Google Scholar). Chinese hamster cells and in cellular as a of the of by and of The used from The heparan sulfate from P. and sulfate sulfate and by 1998; Scholar). and from other from to the DNA binding was by the of or of circular plasmid DNA or forming with of crotamine in The DNA or in with crotamine was with in and was by a fluorescent a to a for from and with a A and The at with crotamine in The was to of made in the or of different of the the was to the and used to the crotamine of with crotamine by the or the protein The the of the the fluorescent was by a with a The of was by as by the and by the which the green fluorescent protein was and of the plasmid DNA for with of crotamine in of with to the and embryonic stem cell in a of mouse embryonic Kerkis Kerkis J. Biol. PubMed Scopus Google Scholar). cells in with and In and In cells used in the in vitro studies cell the cells at a for cell in the of the complexes with or of DNA with of crotamine in of to the cells. 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Biol. PubMed Scopus Google Scholar). cells and the cells with in for at the cells with and to crotamine for different at The fluorescent of with a confocal to complexes by of crotamine with or of the plasmid In this both crotamine in or with the was that of the plasmid DNA to a with of crotamine, a of DNA of The at the was by the in the of circular DNA in A or of the DNA to the of nucleic by the peptide to the of the crotamine and the DNA was for both circular and DNA Moreover, of both crotamine and plasmid DNA or in that crotamine is to with the plasmid DNA as suggested by the in the of the with the of the by the of for DNA and crotamine In by have shown that fluorescent crotamine into the of cells A. Kerkis I. Yamane T. J. 2004; PubMed Scopus Google Scholar). crotamine is to translocate the plasmid DNA into mES cells used in cells have an and are usually a of mouse embryonic which are cells A. Scholar). we also that crotamine in mouse embryonic was to the cytoplasm to translocate into the nucleus of these cells A. Hayashi M.A.F. Yamane Y. Kerkis I. IUBMB Life. 2006; 58: 1-7Crossref PubMed Scopus (74) Google Scholar). mES cell an that for the of the of DNA complexes in both and cells. The of mES was by proliferating cells A and complexes in and with the cells for and A of was used in a cell that is proliferating and of the cells be in at Y. M. J. 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Full Text PDF PubMed Google Scholar). crotamine, the was and the was and the required for the with crotamine, other and the for are in crotamine with a of the of for heparan sulfate and for sulfate and sulfate and heparan sulfate a binding to a by the as E.S. B. J. PubMed Google the crotamine far in the and of The crotamine a at and a at a The in the is with a from residues with with the this from the in the crotamine residues and A. J. 2003; 270: PubMed Scopus Google Scholar, P. T. Yamane T. K. 2005; PubMed Scopus (74) Google Scholar). The by the of are of in the of in a crotamine induced by the of heparan sulfate or sulfate that different are capable of and in the The of the of a crotamine is shown in of the in the and of that was an in the of the or to results demonstrated that with crotamine in the ability of to cell was in and that crotamine is a is that of endocytosis at be to a of the active also to the of the at this T. S. A. A. B. J. Mol. 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