Nuclear Targeting of Macromolecular Polyanions by an HIV-Tat Derived Peptide

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. 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. human immunodeficiency virus Chinese hamster ovary chondroitin sulfate dermatan sulfate glycosaminoglycan heparan sulfate HIV-tat derived peptide proteoglycan protein transduction domain xylosyl transferase deficient heparan sulfate proteoglycans glutathione-S-transferase transactivator of HIV transcription green fluorescence protein fluorescence-activated cell sorter phosphate-buffered saline trans-acting-responsive The plasma membrane defines the border of living cells and constitutes a barrier to extracellular components. Advances in the molecular biology field have resulted in the development of novel therapeutic strategies, e.g. gene therapy and cellular protein delivery, which must rely on the entry of large, polyvalent molecules into the intracellular compartment. Although internalization of DNA has been demonstrated in various cell lines and tissues, it is a relatively inefficient and potentially cytotoxic process (1Wolff J.A. Malone R.W. Williams P. Chong W. Acsadi G. Jani A. Felgner P.L. Science. 1990; 247: 1465-1468Crossref PubMed Scopus (3243) Google Scholar, 2Hengge U.R. Walker P.S. Vogel J.C. J. Clin. Investig. 1996; 97: 2911-2916Crossref PubMed Scopus (175) Google Scholar). Thus, substantial efforts should be focused on mechanistic studies of macromolecular membrane passage, which then form a basis for the construction of novel DNA and protein delivery vehicles. Recently, two major classes of membrane-penetrating proteins have been identified, i.e. arginine/lysine-rich peptides and peptides containing the hydrophobic core region (h region) of signal peptides (see Refs. 3Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 4Hawiger J. Curr. Opin. Chem. Biol. 1999; 3: 89-94Crossref PubMed Scopus (133) Google Scholar for review). Viral proteins, e.g. VP22 and Tat (transactivator of HIV transcription) from herpes simplex and HIV,1 respectively, contain protein transduction domains (PTD) that have been utilized for cellular delivery of fusion proteins or chemically cross-linked cargoes,e.g. the PTD of HIV-Tat displayed on the surface of λ particles facilitated transfer of phage-encapsulated marker genes into mammalian cells (5Eguchi A. Akuta T. Okuyama H. Senda T. Yokoi H. Inokuchi H. Fujita S. Hayakawa T. Takeda K. Hasegawa M. Nakanishi M. J. Biol. 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Biochem. 2001; PubMed Scopus Google Scholar). was from and DNA was from was from and were from and were all from human and all were from and human cells were from the cells were in with and in a cells were in with the same as DNA HS, or was with in for The was then to cell in and the was to for various of or in of cells were with followed by extensive with to extracellular The cells were in and for fluorescence in a with were low in and to extensive with cells were with polyanions for and as in the were then from extracellular by either of the followed by in and on in growth for or with in followed by with In both cells were then in in for were with and and then which is to the intracellular compartment and in a was the of were using a and a confocal was using were in cells per well in growth DNA and the of were by and for The was then to cells that been with the of the in the was and with of growth an of for cells were with and with of cell containing for followed by expression was in of the cell using a was by using cells were with chondroitin and as M. S. Biochem. J. 1999; PubMed Scopus Google Scholar). cell were for with of the containing of or of chondroitin of or was then to the to and respectively, and the was to for the of cells were with of the and Tat internalization was by to the cells were for with with and of to extensive Tat uptake was by cells were with in of for The was then and cell were with to This was followed by of in of was and to to cell was for with or and then to cells for a of Cell was to cell and with and extensive in cell were with of in containing and for was by on and as M. P. Biochem. J. 1999; PubMed Scopus Google Scholar). cell were with either with or for was by on and then as with were to the by the were then for followed by with for and then as was to by chondroitin in or by of with for followed by with as PubMed Scopus Google Scholar). were then to on a as M. M. S. 1996; PubMed Scopus Google Scholar). The was using a in the and is the from two In the were the fluorescence and are of or macromolecular membrane transport in cells, GAG DNA and with the The which and to the PTD of cellular entry of DNA and Chinese hamster ovary cells were with polyanion with or and by of resulted in a to in macromolecular polyanion uptake and of uptake of and DNA was indicating a common uptake of and complexes and We confocal fluorescence to intracellular of complexes. both and uptake of was the and displayed nuclear accumulation an and was In the presence of chains accumulated in and were then to the a gene it was demonstrated that a and in to as with DNA suggesting that DNA to the nuclear compartment of recipient cells The vesicles were and displayed size of both in cells and human in the presence of was from the to cytoplasmic both an and and and This was by confocal of and in in and and a a signal from in the in these conclude that and or DNA are to cytoplasmic vesicles formed de that targets and DNA to the and that macromolecular polyanions cellular uptake of the HIV-Tat transduction nuclear of the the of cytoplasmic vesicles to a was i.e. a that In were with a signal in cells However, addition of complexes with a signal from the indicating rapid of these of complexes to these which to was demonstrated by the confocal of of complexes to vesicles was and of into the The of vesicles was a as by the disappearance of of the complexes to be as to HIV-Tat and its PTD cells via or by as both temperature-dependent A.D. EMBO J. 1991; PubMed Scopus Google Scholar, M. G. A. 2000; PubMed Scopus Google Scholar) and (6Torchilin V.P. Rammohan R. Weissig V. Levchenko T.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8786-8791Crossref PubMed Scopus (707) Google Scholar, E. P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) uptake have been it was that internalization of complexes is a and and and that uptake was that DNA and uptake into endolysosomal vesicles via a and temperature-dependent with were with human cells, and a cell on indicating a role for cell-surface PG in cellular transport of polybasic M. S. Biochem. J. 1999; PubMed Scopus Google Scholar, M. L. L. J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) and full-length HIV-Tat (14Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. 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Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2198) Google Scholar). the it may be that endogenous PG cellular entry of complexes. into the between and endogenous cells were with which is into either in the presence or absence of PG was from cell as well as cell and by In the presence of and of PG to and was We the of full-length HIV-Tat on PG cells were either or with and expression was by In with the of PG to PG was shown in The in be by of on PG by with PG by of of intracellular In the cells were with either in the presence or absence of Cell and cell were by and in the presence of of PG to PG was indicating of complexes from the extracellular In the absence of of PG were internalized and of internalized PG from cells and demonstrated the almost existence of the that with this of efficient uptake of in the presence of was demonstrated by as in Moreover, confocal demonstrated accumulation of after of which is to the with However, after of was retained in and nuclear was after intracellular accumulation of proteoglycans. was as and to cells in the presence or absence of Cell and cell and were and by either on and or on and in was by In and a linear of was of by corresponding to in was on a either or after with chondroitin or after of cells with and for confocal fluorescence of cells with and for or that an HIV-Tat derived peptide targets the macromolecular polyanions DNA and to the nuclear compartment of mammalian cells and that cell-surface PG either or on the of the complexes. this is the to that a peptide from HIV-Tat efficiently via as delivery of covalently attached Because of their DNA and GAG plasma in uptake was demonstrated an ratio of an molecular of and net charge of chain for the in this J. 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