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INTRODUCTION |
Recently, methods have been developed for the delivery of
exogenous proteins into living cells with the help of
membrane-permeable carrier peptides such as
HIV-11 Tat-(48-60) and
Antennapedia-(43-58) (1-11). By genetically or chemically hybridizing
these carrier peptides, the efficient intracellular delivery of various
oligopeptides and proteins was achieved. One of the most amazing
examples is the Tat-
-galactosidase fusion protein (4), which has a
molecular mass as high as 120 kDa. Intraperitoneal injection of the
protein resulted in delivery of the protein with
-galactosidase
activity to various tissues in mice, including the brain. The
peptide-mediated approaches would allow the incorporation of peptides
containing unnatural amino acids or nonpeptide molecules such as
fluorescence probes. These methods would become powerful tools not only
for therapeutic purposes as an alternative to gene delivery, but also
for the understanding of the mechanisms behind fundamental cellular
events, such as signal transduction and gene transcription.
Besides the potential of Tat-(48-60) as a protein carrier, the
internalization mechanism of the peptide attracted our interest. For
example, Tat-(48-60) (GRKKRRQRRRPPQ) is a highly basic and hydrophilic
peptide, which contains 6 arginine and 2 lysine residues in its 13 amino acid residues. However, the peptide was reported to be
translocated through the cell membranes in 5 min at a concentration of
0.1 µM (2). Internalization of the peptide was not
inhibited even at 4 °C. The peptide is less toxic to cells than
other basic membrane-interacting agents. The above features suggested
that the internalization mechanism of Tat-(48-60) was completely
different from the typical transmembrane mechanisms reported so far.
Questions arise as to whether such an efficient translocation is
specific for Tat-(48-60) and Antennapedia-(43-58) peptides and what
is the mechanism of the highly efficient internalization. Based on experiments using synthetic peptides, we suggest the possible presence
of a very similar translocation mechanism to Tat-(48-60) present among
the various arginine-rich peptides. We also suggest the possible
existence of the optimum chain length of arginine peptides for the internalization.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis and Fluorescent Labeling--
All the peptides
used in this study were chemically synthesized by Fmoc
(9-fluorenylmethyloxycarbonyl)-solid-phase peptide synthesis on a Rink
amide resin as reported previously (12). Fluorescent labeling of the
peptides was conducted by the treatment with 1.5 eq of
5-maleimidofluorescein diacetate (Sigma) in dimethylformamide-methanol (1:2) for 3 h followed by reverse-phase HPLC purification. The fidelity of the products was ascertained by time-of-flight mass spectrometry.
Conjugation of Carbonic Anhydrase with Basic
Peptides--
Carbonic anhydrase in phosphate-buffered saline (PBS)
was simultaneously treated with fluorescein-5(6)-carboxamidocaproic acid N-hydroxysuccinimide ester (Sigma) and
N-(6-maleimidocaproyloxy)succinimide ester (Dojin) (15 eq,
each) at room temperature for 1 h to introduce the fluorescein and
the maleimide function to the protein. After the removal of the
unreacted reagents by gel-filtration on a Sephadex G-25 (Amersham
Pharmacia Biotech) column, the cysteine of the respective arginine-rich
peptides was allowed to react with the maleimide moiety on the above
fluorescein-labeled protein at room temperature for 16 h, and then
the unreacted peptides were removed by gel-filtration. Based on the
molecular weight estimation by SDS-polyacrylamide gel electrophoresis,
one or two molecules of basic peptides and fluorescein per protein
were incorporated, respectively.
Cell Culture--
Mouse macrophage RAW264.7 cells were
maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine
serum. Cells were grown on 60-mm dishes and incubated at 37 °C under
5% CO2 to ~70% confluence. A subculture was performed
every 3-4 days.
Peptide Internalization and Visualization--
For each assay,
4 × 104/ml cells were pelleted on a eight-well
Lab-Tek-II chamber slide (Nalge Nunc) (250 µl/well) and cultured for
16 h. After complete adhesion, the culture medium was exchanged. The cells were incubated at 37 °C for 3 h with the fresh medium (250 µl) containing fluorescein-labeled peptides or proteins. The
concentrations of the peptides and proteins were adjusted before
addition to the cell based on their fluorescent intensity. Cells were
washed three times with PBS, fixed with acetone-methanol (1:1) for 2 min at room temperature, washed three times with PBS again, and then
mounted in fluorescent mounting medium containing 15 mM
NaN3 (Dako). The distribution of fluorescein-labeled
peptides was analyzed on a Zeiss Axioskop fluorescence microscope using a 100× oil immersion lens.
Confocal Microscopy--
Cells were grown, incubated with
proteins, and fixed basically as described above. Cells were then
treated with PBS containing 5 µM propidium iodide (200 µl) at room temperature for 30 min, washed four times with PBS, and
mounted in glycerol:PBS (9:1) containing 1%
p-phenylenediamine dihydrochloride. Data were obtained using
a confocal scanning laser microscope MRC 1024 (Bio-Rad) equipped with a
60× oil immersion lens or LSM 510 (Zeiss) equipped with a 40× lens.
MTT Assay--
The MTT assay was conducted basically in the same
manner as reported previously (2). Cells (1 × 104/well) were cultured in 96-microtiter plates in RPMI
1640 medium with 10% heat-inactivated fetal bovine serum in the
presence of peptides (HIV-1 Tat-(48-60): GRKKRRQRRRPPQ-amide;
R9-Tat: GRRRRRRRRRPPQ-amide; HIV-1 Rev-(34-50):
TRQARRNRRRRWRERQR-amide; FHV coat-(35-49): RRRRNRTRRNRRRVR-amide) at
10 or 100 µM. Cells were incubated at 37 °C under 5%
CO2 for 24 h before addition of MTT (Sigma, 5 mg/ml in
PBS) for 4 h. The precipitated MTT formazan was dissolved
overnight in 0.04 N HCl in isopropanol (100 µl). The
absorbance at 570 nm was then measured. Cell viability was expressed as
the ratio of the A570 of cells treated with
peptide over the control samples.
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RESULTS |
Uptake of Tat-(48-60) Analogs by the Macrophage Cell--
To
obtain insight into the translocation mechanisms of the Tat-(48-60)
peptide, Tat-(48-60), its D-amino acid-substituted analog
(D-Tat) and arginine-substituted analog
(R9-Tat), where residues corresponding to positions 49-57
were replaced with arginine, were synthesized (Fig.
1a). An extra cysteine amide
was incorporated into the C terminus of each peptide for the
fluorescent labeling. The peptides corresponding to nuclear
localization sequences (NLS) derived from simian virus 40 (13) and
nucleoplasmin (14) were also synthesized as references. Treatment of
the peptides with 5-maleimidofluorescein diacetate gave the
corresponding fluorescein-labeled peptides. Internalization of the
peptides was monitored by fluorescence microscopic observation after a
3-h incubation of the peptides with mouse macrophage RAW 264.7 cells at
37 °C. As a result, D-Tat and R9-Tat
were internalized into the cell as efficiently as the Tat-(48-60)
peptides, and localization into both the cytoplasm and nucleus was
observed (Fig. 2). A similar
internalization of the D-amino acid analog of Tat was
reported by Huq et al. (15) using a linear peptide
corresponding to residues 37-72. These results would contradict the
idea that a specific receptor may play a crucial role in the
translocation of the Tat-(48-60) peptide. On the other hand, the
simian virus 40-derived and nucleoplasmin-derived peptides showed a
much lower degree of internalization. These NLS-derived peptides are
rich in lysine. The above results suggested that arginine residues
would play an important role in the translocation.

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Fig. 1.
Structure of arginine-rich peptides used in
this study. C-terminal cysteine amide (C*) was
fluorescein-labeled for monitoring the internalization of the peptides
by fluorescence microscopy. D-Amino acids are denoted in
italics. Cysteine residues corresponding to positions 154 and 269 in c-Fos and c-Jun were replaced by serine, respectively.
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Fig. 2.
Translocation of the arginine-rich
Tat-related peptides through the cell membranes. RAW264.7 cells
were treated with fluorescein-labeled peptides derived from HIV-1
Tat-(48-60) (a), R9-Tat (b),
D-Tat (c), and nucleoplasmin-NLS (d)
(10 µM each) for 3 h.
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Translocation of Various Arginine-rich Nucleic Acid-binding
Peptides through the Macrophage Cell Membranes--
Arginine-rich
basic segments are used by a variety of RNA-binding proteins to
recognize specific RNA structures (16). If arginine in the sequence
plays an important role in the translocation, peptides corresponding to
these RNA-binding segments may translocate through the cell membranes.
To test this hypothesis, 10 arginine-rich RNA-binding peptides bearing
a C-terminal Gly-Cys-amide (Fig. 1b) were similarly
prepared, fluorescein-labeled, and applied to the macrophage cells.
To our surprise, all the peptides other than the human U2AF-(142-153)
peptide translocated through the cell membranes and accumulated in the
cytoplasm and nucleus (Fig. 3). As judged
from the fluorescent intensity, the efficiency of incorporation into the cells showed a tendency to correspond to the number of arginine residues in the sequence. Internalization activity of the HIV-1 Rev-(34-50), FHV coat-(35-49), HTLV-II Rex-(4-16), and BMV
Gag-(7-25) peptides, which have more than seven arginine residues in
their sequences, were comparable with that of the Tat-(48-60) peptide. Fluorescence was observed in the cells as early as 5 min after the
addition of these peptides (1 µM) to the medium. Less
extensive internalization was observed in the case of the
N-(1-22),
21 N-(12-29), and yeast PRP6-(129-144) peptides that
have five arginine residues in their sequences. The fluorescent
intensity in the cells treated with the former peptides (0.1 µM) was judged not to be less than that in those treated
with the latter peptides (10 µM). The P22 N-(14-30) and
cowpea chlorotic mottle virus Gag-(7-25) peptides that have six
arginine residues showed a moderate degree of translocation. HIV-1
Tat-(48-60) is reported to translocate through the cell membranes and
accumulate in the nucleus, especially the nucleolus (2). A similar
tendency was observed with the above peptides. Not only the RNA-binding
peptides but also the DNA-binding peptides corresponding to the basic
leucine zipper segments derived from cancer-related proteins, c-Fos and
c-Jun, and the yeast transcription factor, GCN4, which were also rich in arginine (Fig. 1c), were internalized into the cells with
almost the same efficiency as that of Tat-(48-60) (Fig.
4).

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Fig. 3.
Difference in the translocation efficiency in
the arginine-rich RNA-binding peptides after treatment of RAW264.7
cells with the fluorescein-labeled peptides derived from HIV-1
Rev-(34-50) (a), P22 N-(14-30) (b),
and N-(1-22) (c) (10 µM each) for 3 h.
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Fig. 4.
Translocation of DNA-binding peptides through
the cell membranes. RAW264.7 cells were treated with the
fluorescein-labeled peptides derived from human c-Jun-(252-279)
(a) and yeast GCN4-(231-252) (b) (1 µM each) for 3 h.
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HIV-1 Tat-(48-60) was reported to induce little toxicity to HeLa cells
(2). Using R9-Tat, HIV-1 Rev-(34-50), and FHV
coat-(35-49) peptides as representatives of the above arginine-rich
peptides, cytotoxicity of the peptides was investigated. Determined by
the MTT assay, the above peptides did not show a significant
cytotoxicity to the macrophage cells during the treatment with a
peptide (10 µM) for 24 h. At 100 µM,
cell viability of the cells treated with R9-Tat became
70%, whereas viability of those treated with other peptides as well as
HIV-1 Tat-(48-60) was still greater than 95%. These results suggested
that many of the arginine-rich peptides can be of low cytotoxicity as
reported for the HIV-1 Tat-(48-60) peptide.
Consideration of the Translocation Mechanism of the Arginine-rich
Peptides--
The above experiments showed that a variety of
arginine-rich RNA/DNA-binding peptides were able to translocate through
the cell membranes. Little homology in these sequences was observed, except that they all have 5-11 arginine residues. Moreover, the D-amino acid substituted Rev-(34-50) peptide (1 µM) was internalized as efficiently as the
L-peptide in 3 h (data not shown). Circular dichroism
(CD) spectra of the HIV-1 Tat-(48-60), R9-Tat, and FHV coat-(35-49) peptides in methanol were suggestive of their not having
a significant secondary structure (Fig.
5), whereas the HIV-1 Rev-(34-50)
peptide showed a spectrum typical of an
-helical peptide. The U2AF
peptide, which was only slightly internalized into the cell, showed a
spectrum very similar to that of the FHV coat-(35-49) peptide. These
results were suggestive of the absence of even a common secondary
structure in the membrane-permeable peptides. When the cells were
incubated with a peptide (1 µM) at 4 °C for 30 min, no
significant decrease in fluorescent intensity in the cell was observed
using the HIV-1 Rev-(34-50), and FHV coat-(35-49) peptides (Fig.
6). These results suggested that typical endocytosis pathways so far established would not play a crucial role
in the translocation of these arginine-rich peptides.

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Fig. 5.
CD spectra of the arginine-rich peptides in
methanol. HIV-1 Tat-(48-60) (55 µM)
(square), R9-Tat (52 µM)
(diamond), FHV coat-(35-49) (43 µM)
(upward triangle), HIV-1 Rev-(34-50) (39 µM) (circle), and human U2AF-(142-153) (61 µM) (downward triangle).
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Fig. 6.
Effect of temperature on HIV-1 Rev-(34-50)
peptide internalization. The cells were incubated with the peptide
(1 µM) for 30 min at 4 °C or at 37 °C. In the
former case, the cells were preincubated at 4 °C for 1 h before
addition of the peptide. All the following procedures were also
conducted at 4 °C until the completion of the fixation.
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We next focused on the question whether the entry of arginine-rich
peptides into the cells is one-way or not. The cells were treated with
the HIV-1 Rev-(34-50) peptide (1 µM) for 3 h, then the medium was exchanged with a fresh one not containing the peptide. The fluorescence intensity from the cells 1 h later was almost comparable with or only slightly less than that of the cells just before the medium exchange. However, a substantial decrease in the
fluorescence intensity was recognized in the cells 6 h later, and
complete disappearance of the fluorescence was observed 24 h
later. To examine if the above results were due to the leakage of the
peptide from the cells, the medium was analyzed by an HPLC equipped
with a fluorescence spectrophotometer. No peak was detected at the
retention time corresponding to the peptide; however, peaks were
observed that eluted at positions identical with those of the peptide
treated with trypsin (data not shown). Therefore, we concluded that the
decrease in fluorescence intensity of the cells mainly resulted from
the degradation of the peptides, and not from the leakage of the intact
peptide. The question whether the ingested peptide had a certain effect
on the cell growth was also examined. The above HIV-1
Rev-(34-50)-treated cells were harvested 24 h later and counted.
The cell number for the peptide-treated cells was comparable with that
for the control cells (without peptide treatment). Thus, the
peptide-ingesting cells were judged to remain viable to divide with
little effect by the peptide. It would be plausible that the peptide
evenly distributes in each of the daughter cells upon cell division,
since significant differences in the florescence intensity were not
observed among the adjoining cells 6 h later. Considering the
doubling time of the cell, which was estimated to be about 18 h, a
certain amount of cells must have divided within the 6 h. If the
peptides would preferentially stay in one of the daughter cells upon
cell division, a certain discrepancy in the fluorescence intensity will
be observed among the adjacent cells. However, further study will be
necessary to adequately address this question.
Applicability of the Arginine-rich Peptides to the
Intracellular Protein Delivery--
To examine the applicability of
the above basic peptides as protein carriers, we prepared basic
peptide-protein conjugates. Carbonic anhydrase (29 kDa) was selected as
a model protein. Basic peptide-carbonic anhydrase conjugates were
prepared using N-(6-maleimidocaproyloxy)succinimide ester
(EMCS) as a cross-linking agent (17) (Fig.
7A). A fluorescein moiety was
introduced into the protein using the
fluorescein-5(6)-carboxamidocaproic acid
N-hydroxysuccinimide ester simultaneously with EMCS. As
judged from the SDS-polyacrylamide gel electrophoresis of the
conjugates, one to two molecules of the basic peptide and fluorescein
moiety were introduced into a molecule of carbonic anhydrase,
respectively. Carbonic anhydrase was successfully delivered into the
cells with the help of the HIV-1 Rev-(34-50), FHV coat-(35-49), and
R9-Tat peptides as efficiently as with the HIV-1
Tat-(48-60) peptide (Fig. 7B). Accumulation of the
conjugates in the cytosol and nucleus was also observed by fluorescence
microscopy of the cells without fixation (Fig. 7B). Confocal
microscopic analysis of these conjugates demonstrated both cytoplasmic
and nuclear localization and not just attachment to the cellular
membranes (Fig. 7C). On the other hand, fluorescein-labeled
protein without a carrier peptide was located in a limited part of the
cytosol (Fig. 7C). This result suggested that the protein
was captured in the endosomes and was not able to be released into the
cytosol. Myoglobin (17 kDa) was also introduced into the cell with the
help of these carrier peptides (data not shown).

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Fig. 7.
Delivery of carbonic anhydrase into RAW264.7
cells with the help of arginine-rich basic peptides. A,
schematic representation of the conjugates. B,
fluorescence microscopy of the cells treated with carbonic anhydrase
conjugated with the HIV-1 Rev-(34-50) (a), FHV
coat-(35-49) (b), and HIV-1 Tat-(48-60) (c)
peptides (1 µM each) for 3 h, respectively.
Accumulation of the HIV-1 Rev-(34-50)-carbonic anhydrase conjugate in
the cytosol and nucleus was also observed by the fluorescence
microscopy of the cells without fixation (protein concentration: 10 µM) (d). C, confocal microscopic
observation of the cells treated with carbonic anhydrase conjugated
with the HIV-1 Rev-(34-50) peptide (1 µM) with nucleus
staining by propidium iodide (PI) (a). The
protein without the carrier peptides (1 µM) did not show
a significant accumulation in the nucleus (b).
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Effect of the Length of Arginine Chain on the
Internalization--
The above data strongly suggested the importance
of arginine residues in the internalization. The possible existence of
the unique internalization mechanism common in these arginine-rich peptides was also suggested. We then examined the effect of the number
of arginine residues in the sequences. For simplification, peptides
that are composed of 4-16 residues of arginine were prepared (Fig.
1d). To their C termini, the Gly-Cys-amide segment was also attached for the fluorescein labeling. These results are shown in Fig.
8A. Considerable difference
was recognized on the translocation efficiency and intracellular
localization among these peptides. R4 showed extremely low
translocation activity. R6 and R8 exhibited the
maximum internalization and accumulation in the nucleus. What is
interesting is that the degree of internalization decreased as the
chain length further increased. For the R16,
internalization of the peptide was not significant. The same kind of
difference was recognized in the experiments using the conjugates of
carbonic anhydrase with the arginine peptides (Fig. 8B). A
similar tendency was observed on the protein delivery using
R8 and R16 as the carrier molecules. Based on
the confocal laser microscopic observations, the
R8-carbonic anhydrase conjugate was efficiently
internalized into the macrophage cells and accumulation in the nucleus
was observed as was seen in the case of the HIV-1 Rev-(34-50)
conjugate. In contrast, the R16-conjugate seemed to mainly
reside on the cell membranes after a 3-h incubation with the conjugate,
but significant accumulation in the nucleus was not observed.

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Fig. 8.
Fluorescence microscopic observation of the
cells treated with polyarginine peptides (1 µM) for 3 h (A),
and confocal microscopic observation of the cells treated with carbonic
anhydrase conjugated with the R8 or R16
peptides (1 µM) with nucleus
staining by propidium iodide (PI)
(B).
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DISCUSSION |
In this report, we have shown that not only Tat-(48-60) but also
various arginine-rich peptides were able to translocate through the
mouse macrophage membranes. These peptides include the D- and arginine-substituted HIV-1 Tat-(48-60) analogs, RNA-binding peptides derived from proteins, such as HIV-1 Rev, HTLV-II Rex, BMV
Gag, and FHV coat proteins, and the DNA-binding segments from c-Fos,
c-Jun, and the GCN4 proteins. There seems a common or very similar
mechanism for the internalization among these peptides. The mechanism
is explained neither by adsorptive-mediated endocytosis nor by
receptor-mediated endocytosis because the peptides were internalized by
the cell at 4 °C, and there seemed little homology both in the
primary and secondary structures among these membrane-permeable peptides except that they have several arginine residues in the sequences. These results strongly suggest the possible presence of the
common and undefined internalization mechanisms ubiquitously laying
among the arginine-rich basic peptides. As one more new finding
concerning the features of the internalization, we have shown that the
number of arginine residues has a significant influence on the method
of internalization and that there seems to be an optimal number of
arginine residues for the internalization. There still remain questions
why such efficient translocation is possible for the arginine-rich
peptides. Possible hydrogen bond formation of arginine with lipid
phosphates (18) or interaction with extracellular matrices such as
heparan sulfate (19) may be involved in the initial step during the
mechanism. However, as was seen in the case of the R16
peptide, it is not enough to explain the mechanism only by considering
adsorption of peptides on the membranes.
Tat-(48-60) has been reported to carry various proteins into the cells
not only into cultured cells but also into the various organs of a
living mouse (4). As the arginine-rich peptides examined here seem to
have a similar ability as carriers of proteins, further study of the
arginine-based peptides may result in finding peptides penetrating to
some specific cells by themselves or with the help of other address peptides.
The results obtained here not only shed light on the possible presence
of new types of ubiquitous transmembrane mechanisms for the
arginine-rich peptides, but also on the development of novel carrier
molecules for the intracellular protein delivery.