Division of Life Sciences, Hallym University, Chunchon 200-702, Korea1
Department of Physiology, College of Medicine, Hallym University, Chunchon 200-702, Korea2
Author for correspondence: Soo Young Choi. Present address: Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea. Fax +82 33 241 1463. e-mail sychoi{at}hallym.ac.kr
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Abstract |
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Introduction |
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Human immunodeficiency virus type 1 (HIV-1) Tat protein, which can be secreted from infected cells, has the ability to enter neighbouring cells through the plasma membrane and accumulate in the cell (Frankel & Pabo, 1988 ; Ensoli et al., 1993
; Green & Loewenstein, 1988
; Mann & Frankel, 1991
). Due to this property, whole Tat protein or part of it has been tested because of its ability to deliver several proteins, including ovalbumin,
-galactosidase and horseradish peroxidase into cells (Fawell et al., 1994
; Watson & Edwards, 1999
). A basic domain of the Tat protein rich in arginine and lysine residues, called the protein transduction domain (PTD), has been identified as being responsible for the ability to traverse the plasma membrane. It has also recently been shown to serve as a carrier to direct the uptake of heterologous proteins into cells by generating genetic in-frame PTD fusion proteins (Jin et al., 2001
; Kwon et al., 2000
; Nagahara et al., 1998
; Vocero-Akbani et al., 1999
). Furthermore, it has recently been reported that
-galactosidase, fused to the basic domain of HIV-1 Tat, was delivered in the biologically active form to all tissues including the brain when injected intraperitoneally into mice (Schwarze et al., 1999
).
Other proteins, such as the homeodomain of Drosophila antennapedia (Antp) and the herpes simplex virus type 1 VP22 transcription factor, have also been shown to enter cells in culture (Joliot et al., 1991 ; Elliott & OHare, 1997
; Han et al., 2000
). Small regions of these proteins, ranging from 11 to 34 amino acids in length, were able to cross the lipid bilayer of cells either alone or fused to various polypeptides or oligonucleotides (reviewed in Kwon et al., 2000
; Schwarze & Dowdy, 2000
; Schwarze et al., 2000
; Lindgren et al., 2000
; Derossi et al., 1998
).
Although it has been suggested that transduction occurs in a receptor- and transporter-independent fashion that appears to target the lipid bilayer directly, the mechanism of transduction mediated by HIV-1 PTD needs to be elucidated (Derossi et al., 1996 ; Vives et al., 1997
). The cellular internalization of homeoproteins and homeodomains occurs at 4 °C and 37 °C and cannot be saturated, which suggests a transduction mechanism that is energy- and receptor-protein-independent (Joliot et al., 1991
; Derossi et al., 1994
, 1996
). Therefore, transduction by these proteins is independent of the classical endocytosis pathway, and it shows such characteristics as high efficiency, non-cell-type specificity and low toxicity.
In the present study, we have defined the sequence requirements for the HIV-1 PTD by deletion analysis and described the contribution of basic amino acid sequences to transduction activity by substitution of HIV-1 PTD with nine consecutive arginines or lysines.
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Methods |
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pTatGFP was constructed in the following manner to express the basic domain (amino acids 4857) of HIV-1 Tat as a fusion protein with GFP. First, two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide, encoding the nine amino acids from the basic domain of HIV-1 Tat. The sequences were (top strand) 5' TAGGAAGAAGCGGAGACAGCGACGAAGAC 3' and (bottom strand) 5' TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC 3'. The double-stranded oligonucleotide was directly ligated into the NdeIXhoI-digested pGFP, in-frame with the 6-His open reading frame to generate the TatGFP expression plasmid, pTatGFP. This plasmid, encoding the basic domain (amino acids 4857) of HIV-1 Tat fused with GFP, was modified such that oligonucleotides corresponding to a series of PTD variants were annealed and inserted between the His tag region and the GFP gene of pGFP. The sequences of the oligonucleotides cloned into the plasmid were confirmed using a fluorescence-based automated sequencer (model 373A; Applied Biosystems). The oligonucleotides used in the construction of the plasmids with a series of PTD variants, the corresponding amino acids and the resulting GFP fusion proteins are summarized in Table 1.
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Cell culture and transduction of GFP fusion proteins.
HeLa cells were cultured in Dulbeccos modified Eagles medium containing 20 mM HEPESNaOH, pH 7·4, 5 mM NaHCO3, 10% foetal bovine serum (FBS) and antibiotics (100 µg/ml streptomycin, 100 U/ml penicillin) at 37 °C. For the transduction of GFP fusion proteins, cells were grown to confluence in six-well plates. The culture medium was replaced with fresh medium containing 10% FBS and was then treated with various concentrations of GFP fusion proteins for indicated time intervals. The cells were washed with PBS, followed either by an acid wash with 0·2 M glycineHCl, pH 2·2, or by trypsinization with trypsin without EDTA (Gibco) for 10 min and then washed with PBS. Cells were prepared for analysis by Western blot or confocal microscopy as described below.
Subcellular fractionation of the transduced cells.
The nuclear and cytosolic fractions were prepared as previously described by Nare et al. (1999) . Briefly, transduced HeLa cells (
5x106) were washed with PBS, acid-washed with 0·2 M glycineHCl, pH 2·2, and trypsinized for 10 min at 37 °C. The cells were harvested after washing with cold PBS and pelleted. The cells were then resuspended in 1 ml of NP-40 buffer (0·01 M TrisHCl, 0·01 M NaCl, 0·003 M MgCl2, 0·03 M sucrose, 0·1 mM PMSF, 0·5% NP-40) by gentle pipetting and incubated on ice for 10 min. Cells were spun through a sucrose cushion at 1000 g for 10 min and the cytosolic fractions were collected from the supernatants. Pellets were washed with 1 ml NP-40 buffer to completely remove cytosolic fractions. The nuclei were lysed in a lysis buffer (50 mM TrisHCl, pH 8·0, 150 mM NaCl, 0·02% sodium azide, 100 µg/ml PMSF, 1% Triton X-100). The resulting nuclear and cytosolic lysates were analysed by Western blotting.
Western blot analysis.
Cell lysates were prepared by lysing monolayer cells on a six-well plate with a lysis buffer (125 mM TrisHCl, pH 6·8, 2% SDS, 10%, v/v, glycerol). For Western blot analysis, 15 µg of the protein from each whole cell lysate was run on a 12% SDSpolyacrylamide gel. Proteins were electrotransferred to a nitrocellulose membrane, which was then blocked with 10% (w/v) dry milk in PBS. The membrane was probed with rabbit anti-GFP polyclonal antibody (Clontech) diluted 1:1000, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (Sigma) diluted 1:10000. The bound antibodies were visualized by enhanced chemiluminescence (ECL; Amersham) as recommended by the manufacturer (Kwon et al., 2000 ). The same membrane was stripped and reprobed with an anti-actin antibody (cytosolic marker) (Oncogene) or an anti-poly(ADPribose) polymerase (PARP) antibody (nuclear marker) (Biomol. Plymouth Meeting, PA).
Analysis of transduced cells by fluorescence microscopy and confocal microscopy.
HeLa cells grown on coverslips to 5070% confluency were treated with various amounts of GFP fusion proteins. Following incubation for 1 h, the cells were washed twice with PBS, trypsinized and then fixed in 3·7% (v/v) formaldehyde in PBS for 5 min at room temperature. The cells were washed again with PBS before being mounted in PBS containing 90% glycerol and 0·1% phenylenediamine. The distribution of the fluorescence was analysed on an Olympus epifluorescence microscope with a 488 nm fluorescent filter (Lee et al., 1999 ). The transduction of GFP fusion proteins into the cells was also confirmed using confocal microscopy. The transduced cells were washed twice with PBS, and then acid-washed with 0·2 M glycineHCl, pH 2·2 at room temperature. Cells were fixed in 3·7% (v/v) formaldehyde in PBS for 5 min at room temperature and stained for 30 min with 2 µg/ml propidium iodide (PI) (Sigma) to visualize the nuclei. The fixed cells were transferred to a chamber on the stage of a Zeiss Axiovert S100 microscope and observed using a confocal laser-scanning system (Bio-Rad MRC-1024ES). Wavelengths of 535 nm and >395 nm were used to excite PI and GFP, respectively; emission spectra were collected with 617 nm and 540 nm bandpass filters. The fluorescence images of the cells transduced with GFP fusion proteins were recorded every 0·25 s (magnification x640).
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Results |
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To construct a vector for expression of TatGFP fusion protein, the initial coding sequence of GFP was amplified by PCR and inserted into a pET15b vector using XhoI and BamHI sites. Next, two oligonucleotides encoding the full length of the HIV-1 Tat PTD (amino acids 4857) were inserted into the constructed vector to generate TatGFP expression vectors. Thus, the constructed pTatGFP vector encoded GFP as a fusion protein with HIV-1 PTD (Fig. 1A). Expression vectors containing deleted or substituted PTDs were constructed by the same method (Fig. 1A
, Table 1
).
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To investigate whether or not the denatured Tat(4857)GFP was transduced and restored to biological activity in the cells, various concentrations of denatured Tat(4857)GFP protein were added to the culture media of HeLa cells for 1 h, and the protein levels and fluorescence intensity were analysed by Western blot analysis and fluorescence microscopy, respectively. As shown in Fig. 2(A), the level of transduced protein inside the cells increased and fluorescence signals were detected in a dose-dependent manner (Fig. 2B
). Unlike TatGFP, GFP without PTD could not be transduced into the cells. These data indicate that the denatured Tat(4857)GFP was transduced and correctly refolded into a biologically active conformation in the mammalian cells.
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The transduction activity of Tat(4957)GFP was indistinguishable from that of Tat(4857)GFP (Fig. 3A, compare lanes 9 and 10), whereas Tat(5057)GFP and Tat(5157)GFP appeared to be transduced at slightly lower levels than Tat(4857)GFP (Fig. 3A
, compare lanes 2, 8 and 9). These results suggested that deletion of the amino acid residue corresponding to Gly-48 did not affect the transduction efficiency, and that the minimum basic domain sequences for the efficient transduction was from 49 to 57 in the PTD. Further deletion of Lys-50 resulted in a slight decrease of transduction activity, while deletion of Lys-51 dramatically abolished transduction activity (Fig. 3A
, B
). In a similar manner, sequential deletions of the C-terminal amino acids (Arg-57, Arg-56, Arg-55 and Gln-54) produced lower efficiencies of transduction activity. The results analysed by fluorescence microscopy were consistent with those transduction activities shown in the Western blot analysis (Fig. 3C
). In general, TatGFP fusion protein with a full-sized basic domain was translocated into both the nucleus and the cytoplasm, while GFP fusion proteins with a truncated basic domain were present in the cell at a low level. Therefore, we concluded that the basic domain from 49 to 57 is required for efficient transduction activity, whereas further deletions of amino acids from the N or C terminus of the Tat basic domain lead to significant loss of transduction activity.
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To determine the transduction activity of Tat(4957)GFP, 9LysGFP and 9ArgGFP, various concentrations of each fusion protein were added to the culture media of HeLa cells for 1 h and transduction analysed by Western blotting and fluorescence microscopy. As shown in Fig. 4, all GFP fusion proteins were successfully delivered into the cells with similar transduction efficiency, whereas control GFP was not delivered into the cells. Transduced proteins appeared to locate either in the cytoplasm or in the nucleus. To clarify further the subcellular localization of transduced proteins in the cells, nuclear and cytosolic fractions were prepared from cells transduced with Tat(4957)GFP, 9LysGFP and 9ArgGFP and analysed by Western blotting using anti-GFP, anti-actin or anti-PARP antibodies. As shown in Fig. 5
, Tat(4957)GFP, 9LysGFP and 9ArgGFP were detected at similar intensity in the nucleus as well as in the cytoplasm of transduced cells, whereas control GFP was not detected in the cells. When the intracellular localization of transduced proteins was visualized by confocal microscopy, Tat(4957)GFP, 9LysGFP and 9ArgGFP were found to be present in both the nucleus and the cytosol (Fig. 6
). The distribution of 9LysGFP and 9ArgGFP was very similar to that of Tat(4957)GFP, although neither polylysine nor polyarginine contains a typical nuclear-localization signal. To confirm that Tat(4957)GFP, 9LysGFP and 9ArgGFP can localize to the nucleus, transduced cells were double-stained with the nucleus-specific marker PI. Dual-colour detection of the various GFP fusion proteins and PI demonstrated that Tat(4957)GFP, 9LysGFP and 9ArgGFP localized to the nucleus, while GFP alone did not (Fig. 6
). Taken together, these results indicated that substituting the HIV-1 basic domain with nine arginines or nine lysines retained full transduction activity and did not affect the subcellular localization of transduced proteins.
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Discussion |
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It has been suggested that the transduction of protein into cells could be mediated by the HIV-1 basic domain due simply to an inherent characteristic of specific amino acid sequences such as arginine or lysine (Schwarze et al., 2000 ). This hypothesis is supported by our result with substitutions of the HIV-1 basic domain by nine arginine or nine lysine residues. The substituted 9ArgGFP and 9LysGFP were each shown to transduce as efficiently into cells as TatGFP. When nine arginine or nine lysine residues were fused with other heterologous proteins, such as superoxide dismutase (Park et al., 2002
), catalase (Jin et al., 2001
), E. coli nitroreductase and p21CIP1/WAF1 (unpublished results), these fusion proteins were also efficiently transduced into cells. These homogeneous polyarginine and polylysine peptides may be non-immunogenic when administered in vivo. Therefore, the protein-transduction domains substituted with arginines or lysines as the sole carrier vehicle may have advantages over HIV-1 PTD itself.
Recently, Wender et al. (2000) examined the transduction efficiency of truncated versions of the Tat basic domain and of nine arginine peptides that were directly fluorescent-labelled. However, in the present study, the potential of various oligopeptides as PTDs was studied as forms genetically fused to heterologous protein. This approach may more closely reflect eventual pharmaceutical usage for protein delivery. We found that the relative transduction efficiencies of fused proteins with different length of PTD were similar to those of free oligopeptides with a corresponding number of basic residues, although there were some differences in the degree of loss between the free and the fused forms. We found that 9ArgGFP was transduced into the cells with a similar efficiency to Tat(4957)GFP, while Wender et al. (2000)
observed that a fluorescent-labelled nine-arginine peptide was more efficient at entering cells than Tat(4957) (RKKRRQRRR). This discrepancy in the transduction efficiency may derive from the properties of the fusion target protein, including the degree of unfolding, polarity and molecular shape of the protein.
When the TatGFP fusion proteins were observed by fluorescence microscopy and confocal microscopy, they were translocated into both the cytoplasm and the nucleus. It has been suggested that this cluster of nine basic amino acids, Tat(4957), serves as a nuclear-localization signal (NLS) (Efthymiadis et al., 1998 ; Ruben et al., 1989
). Although the glycine residue at the 48 position was initially reported to serve as part of the NLS, it is clear that this residue is required for neither transduction nor nuclear localization. This was demonstrated by Tat(4957)GFP, which contained no glycine residue, being transduced into cells and localized both in the cytoplasm and the nucleus with an efficiency similar to that of Tat(4857)GFP (Fig. 5
). Like Tat(4957)GFP, the translocalization of 9LysGFP and 9ArgGFP was also observed in the nucleus, as well as in the cytosol (Fig. 6
).
The molecular mechanisms of the transduction of heterologous protein mediated by the HIV-1 basic domain through the cell membrane are not yet characterized. In general, the denatured proteins containing HIV-1 Tat PTD transduce more efficiently into cells than do correctly folded proteins (Kwon et al., 2000 ; Nagahara et al., 1998
; Schwarze et al., 1999
). It has been suggested that the increased transduction efficiency of denatured proteins may result from reduced structural constraints, which allow proteins to pass more easily through the cell membrane (Nagahara et al., 1998
). Supporting evidence comes from a previous study, which found that unfolding was necessary to traverse the cellular membrane when Tat was used to ferry dihydrofolate reductase into cells (Bonifaci et al., 1995
). Further evidence has been provided by a recent study on the structural characterization of HIV-1 Tat showing that the basic region of HIV-1 Tat is well exposed to solvent (Peloponese et al., 2000
). Therefore, these results imply that the conformation of proteins may be critical in the efficient transduction of heterologous proteins into cells.
In summary, GFP fusion proteins containing the various protein-transduction domains developed in this study facilitated a comparative analysis of transduction efficiency and provided useful tools for studying the mechanism of transduction mediated by the basic domain of the HIV-1 Tat protein. In addition, these GFP fusion proteins have the potential to be used in a variety of applications, including studies of in vivo and in vitro protein delivery.
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Acknowledgments |
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References |
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Received 16 November 2001;
accepted 4 January 2002.