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J. Biol. Chem., Vol. 282, Issue 29, 21349-21360, July 20, 2007

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

Fábio Dupart Nascimento¹, Mirian A. F. Hayashi², Alexandre Kerkis^¶, Vitor Oliveira^||3, Eduardo B. Oliveira^**, Gandhi Rádis-Baptista, Helena Bonciani Nader, Tetsuo Yamane, Ivarne Luis dos Santos Tersariol^¶¶4, and Irina Kerkis^||||5

From the Departamento de Bioquímica, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua 3 de Maio, 100, Ed. INFAR, CEP 04044-020, São Paulo, Centro de Toxinologia Aplicada, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900, São Paulo, ^¶Clínica e Centro de Pesquisa em Reproduão Humana Roger Abdelmassih Av. Brasil, 1085, CEP 01431-000, São Paulo, ^||Laboratório de Neurociências, Universidade Cidade de São Paulo Rua Cesário Galeno, 448, CEP 03071-000, São Paulo, ^**Departamento de Bioquímica e Imunologia, Universidade de São Paulo Av. Bandeirantes, 3900, CEP 14049-900, Ribeirão Preto, Centro de Ciências Biológicas, Universidade Federal de Pernambuco Cidade Universitária, CEP 50732-970, Recife, Pernambuco, Instituto de Pesquisas Energéticas Nucleares, Universidade de São Paulo Av. Prof. Lineu Prestes, 2242, CEP 05508-000, São Paulo, ^¶¶Centro Interdisciplinar de Investigaão Bioquímica, Universidade de Mogi das Cruzes, Av. Dr. Candido Xavier de Almeida Souza, 200, CEP 08780-210, Mogi das Cruzes, SP, and ^||||Laboratório de Genética, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900, São Paulo, Brazil

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.

Received for publication, May 22, 2006 , and in revised form, March 8, 2007.

^* This study was supported by grants from Fundaão de Amparo à Pesquisa do Estado de São Paulo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Med Discovery S.A., Ch. des Aulx 16, zip code 1228, Geneva, Switzerland.

² Present address: Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio, 100, 04044-020, São Paulo, SP, Brazil.

³ Present address: Departamento de Biofísica, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio, 100, 04044-020, São Paulo, SP, Brazil.

⁴ To whom correspondence may be addressed: Centro Interdisciplinar de Investigaão Bioquímica, Universidade de Mogi das Cruzes, Prédio I, sala 1S-15, Av. Dr. Candido Xavier de Almeida Souza, 200, CEP 08780-210, Mogi das Cruzes, SP, Brazil. Fax: 55-11-4798-7102; E-mail: ivarne{at}umc.br. ⁵ To whom correspondence may be addressed: Laboratório de Genética, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil. Tel.: 55-11-3726-7222 (ext. 2287); E-mail: ikerkis{at}butantan.gov.br.

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