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Originally published In Press as doi:10.1074/jbc.M205395200 on August 5, 2002

J. Biol. Chem., Vol. 277, Issue 41, 38877-38883, October 11, 2002
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Nuclear Targeting of Macromolecular Polyanions by an HIV-Tat Derived Peptide
ROLE FOR CELL-SURFACE PROTEOGLYCANS*

Staffan Sandgren, Fang Cheng, and Mattias BeltingDagger

From the Department of Cell and Molecular Biology, Section of Cell and Matrix Biology, Lund University, BMC, C13, S-221 84 Lund, Sweden

New therapies based on gene transfer and protein delivery require a better understanding of the basic mechanisms of macromolecular membrane transport. We have studied cellular uptake of macromolecular polyanions, i.e. DNA and glycosaminoglycans, and a polybasic HIV-Tat derived peptide (GRKKRRQRRRPPQC) using fluorescence assisted cell sorting and confocal fluorescence microscopy. The transactivator of HIV transcription (Tat) peptide stimulated cellular uptake of both DNA and heparan sulfate in a time-, concentration-, and temperature-dependent manner. Peptide-polyanion complexes accumulated in large, acidic, cytoplasmic vesicles formed de novo. This was followed by transfer of polyanion into the nuclear compartment and subsequent disappearance of the endolysosomal vesicles. In the absence of polyanion the Tat peptide displayed rapid accumulation in the nuclear compartment. However, in the presence of polyanion the peptide was almost exclusively retained in cytoplasmic vesicles. Cell-surface proteoglycans played a pivotal role in the uptake of complexes exhibiting a relatively high peptide to polyanion ratio, corresponding to a net positive charge of the complexes. Uptake of polyanions per se or complexes with a relatively low peptide to polyanion ratio was favored by proteoglycan deficiency in the recipient cells, indicating the existence of distinct transport mechanisms. Moreover, expression of full-length HIV-Tat as well as exogenous addition of HIV-Tat peptide resulted in cellular accumulation of endogenous proteoglycans. We conclude that an HIV-Tat derived peptide efficiently targets extraneous DNA and glycosaminoglycans to the nuclear compartment and that proteoglycans serve a regulatory role in these processes, which may have implications for directed gene and drug delivery in vivo.

* This work was supported by grants from the Glycoconjugates in Biological Systems Programme of the Strategic Research Fund (to F. C.), the Swedish Cancer Fund (to S. S.), the Crafoord Foundation, the Royal Physiographic Society of Lund, the Zoégas Foundation, the Swedish Society for Medical Research, and the Medical Faculty at Lund University (to M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Section of Cell and Matrix Biology, BMC, C13, S-221 84, Lund, Sweden. Tel.: 46-46-2224077; Fax: 46-46-2223128; E-mail: Mattias.Belting@medkem.lu.se.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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