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Title: Possible existence of common internalization mechanisms among arginine-rich
peptides*
Authors: Tomoki Suzuki, Shiroh Futaki,§ Miki Niwa, Seigo Tanaka, Kunihiro Ueda,
and Yukio Sugiura
Institution: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011,
Japan
Running title: common internalization mechanisms of arginine-rich peptides
§Corresponding author:
Shiroh Futaki, Ph. D.
Associate Professor
Institute for Chemical Research, Kyoto University
Uji, Kyoto 611-0011, Japan
Phone: +81-774-38-3211; fax +81-774-32-3038
E-mail [email protected]
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SUMMARY
Basic peptides such as HIV-1 Tat-(48-60) and Drosophila Antennapedia-(43-58) have
been reported to have a membrane permeability and a carrier function for intracellular protein
delivery. We have shown that not only Tat-(48-60) but many arginine-rich peptides, including
HIV-1 Rev-(34-50), flock house virus (FHV) coat protein-(35-49) as well as octaarginine (Arg)8,
efficiently translocated through the cell membranes and worked as protein carriers [Futaki et al.,
(2001) J. Biol. Chem. 276, 5836]. Quantification and time-course analyses of the cellular uptake
of the above peptides by mouse macrophage RAW264.7, human cervical carcinoma HeLa and
simian kidney COS-7 cells revealed that Rev-(34-50) and (Arg)8 had a comparable translocation
efficiency to Tat-(48-60). Internalization of Tat-(48-60) and Rev-(34-50) was saturable and
inhibited by the excess addition of the other peptide. Typical endocytosis and metabolic
inhibitors had little effect on the internalization. The uptake of these peptides was significantly
inhibited in the presence of heparan sulfate or chondroitin sulfates A, B, and C. Treatment of the
cells with the anti-heparan sulfate antibody or heparan sulfate lyase III (heparinase III) also
lowered the translocation of these peptides. These results strongly suggest that the arginine-rich
basic peptides share a certain part of the internalization pathway and that the interaction with
sulfated glycosaminoglycans on the cell surface may contribute to the initial stage of the
internalization.
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INTRODUCTION
Basic peptides derived from the HIV1-1 Tat protein [Tat-(48-60)] and Drosophila
Antennapedia protein [Antp-(43-58)] have been reported to have the ability to translocate
through the cell membranes and to carry exogenous molecules into the cytoplasm and nucleus
(1-13). A 119 kDa protein, β-galactosidase, genetically fused with the former peptide segment,
was successfully carried into various tissues in mice including the brain via intraperitoneal
injection (6). The X-gal staining of the tissues indicated that the fusion protein was delivered in
its active form. OligoDNAs and metal chelates were also brought into cells using the Tat
derived peptide (4, 7). Such a method to deliver bioactive molecules into cells using membrane
permeable peptides has a great potential for therapeutic fields.
We have recently demonstrated that not only Tat-(48-60) and Antp-(43-58), but also
various arginine-rich RNA or DNA-binding peptides such as HIV-1 Rev-(34-50) and flock
house virus (FHV) coat-(35-49) were membrane permeable and have the ability to bring
exogenous protein into cells (14). Even octaarginine (Arg)8 gave similar results based on the
fluorescence microscopic observation of the fluorescein-labeled peptides (14, 15). These
peptides seem to have other similarities in translocation, namely, facile internalization within 5
min, little uptake inhibition at 4 °C, and localization in the nucleus and cytosol. The above
results suggested the possible existence of an ubiquitous mechanism for the internalization of
the arginine-rich peptides. As there were no sequence similarities among these peptides except
that they had several arginine residues, arginine seemed to be the key amino acid for the
membrane permeability.
In spite of the great potential of the arginine-rich peptides as carriers of proteins,
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nucleic acids, and other bioactive compounds, little is known about the mechanism of their
internalization. Involvement of the cell-surface heparan sulfate (HS) and low-density lipoprotein
receptor-related protein (LRP) was suggested in the translocation of the full length Tat protein
(16, 17). The addition of HS and the inhibitor of LRP to the culture medium produced a
significant decrease in the cellular uptake of the protein. However, the uptake of the full length
Tat protein suffered a certain decrease at 4 °C (16), and some energy-dependent endocytosis
pathway seemed to play a significant role in the internalization of the Tat protein. These results
suggested that the mechanisms of internalization of the Tat-(48-60) peptide and the full-length
Tat protein may not be completely parallel. Actually, importance of the “core” domain [Tat-(37-
48)] of Tat protein has been claimed for the LRP-dependent internalization pathway (16).
We have pointed out that many arginine-rich peptides showed very similar
characteristics in translocation with HIV-1 Tat-(48-60) (14). Not to mention the translocation
mechanisms of these arginine-rich peptides, it is also unclear whether these peptides share the
common pathway for internalization. In this study, we conducted the quantification of the
cellular uptake of these arginine-rich peptides in order to compare their translocation efficiency
using the RAW264.7, HeLa, and COS-7 cell lines. We also examined whether the
internalization of a peptide could be competitively inhibited in the presence of other basic
peptides. Finally, to interpret the internalization mechanism, we examined the effect of the cell
surface sulfated polysaccharides as well as endocytosis and metabolic inhibitors.
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MATERIALS AND METHODS
Peptide synthesis and fluorescein or rhodamine labeling
All the peptides used in this study were chemically synthesized by Fmoc (9-
fluorenylmethyloxycarbonyl)-solid-phase peptide synthesis on a Rink amide resin and
fluorescein labeling was conducted using 5-maleimidofluorescein diacetate (Sigma Chemical,
St. Louis, MO) as previously reported (14, 18). Rhodamine labeling of the peptides was
conducted by the treatment of the peptides with 1.5 eq. of tetramethylrhodamine-5-maleimide
(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.
Cell culture
HeLa cells were purchased from the Riken Gene Bank (Tsukuba, Japan) and cultured in alpha-
minimum essential medium (α-MEM, purchased from GIBCO Life Technologies, Grand Island,
NY) supplemented with 10% (v/v) calf serum (GIBCO) without antibiotics. Cells were grown
on 100 mm dishes in an atmosphere of 5% CO2 at 37 °C. COS-7 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, purchased from Nissui Pharmaceutical, Tokyo,
Japan) supplemented with 10% (v/v) fetal bovine serum (Trace Scientific, Melbourne,
Australia) without antibiotics. The sub-culture was conducted every 3-4 days using the cells
grown to sub-confluence. RAW264.7 cells were cultivated as previously reported (14).
Peptide internalization and visualization
HeLa cells were seeded on a eight-well Lab-Tek-II chamber slide (Nalge Nunc International,
Naperville, IL) at a density of 1 × 104 cells per well in α-MEM containing 10% calf serum. To
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investigate the saturation of the internalization of the arginine-rich peptides, cells were sub-
cultured on a chamber slide and incubated for 48 h, then the medium was replaced by fresh
medium. An excess amount (100 µM) of non-labeled arginine-rich peptide was added to
medium, followed by the addition of the fluorescein-labeled arginine-rich peptide (1 µM). After
a 3-hour incubation, the cells were washed three times with ice-cold phosphate-buffered saline
(PBS), fixed by acetone-methanol (1:1) for 1 min at room temperature, washed with PBS again,
and then mounted with 0.01% w/v p-phenylenediamine dihydrochloride in glycerol.
Treatment of the cells with sulfated polysaccharides [heparan sulfate (HS), chondroitin
sulfate (CS) A, B, and C (Sigma)], heparinase III (Sigma) or anti-HS antibody (Seikagaku Corp.
Tokyo, Japan) was conducted under serum-free conditions. The HeLa cells treated with one of
the above compounds for 30 min were incubated with a fluorescein-labeled arginine-rich
peptide. After an additional 30-minute incubation, washing and fixation were conducted as
described above. The intracellular distribution of labeled peptides was observed using a
fluorescence microscope IX-70 (Olympus) equipped with a 40 × lens.
Quantification of internalized peptides
For the quantification experiment, HeLa, COS-7 and RAW264.7 cells were seeded on a 35-mm
culture dish at a density of 1 × 105 cells per dish. After a 48-hour incubation, the medium was
changed using fresh medium containing 1 µM of a rhodamine-labeled arginine-rich peptide.
Cells were incubated with the peptide-containing medium for 3 h, then washed three times with
ice-cold PBS and lysed with PBS containing 0.5% Triton X-100. The cell lysate was centrifuged
and the fluorescence intensity of the supernatant was measured.
Treatment of the HeLa cells with various inhibitors [brefeldin A, colchicine, nystatin,
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taxol, and wortmannin (Wako Pure Chemicals, Osaka, Japan), chloroquine, cytochalasin D,
nocodazole, rotenone, and sodium azide (Sigma)] was conducted in serum-free α-MEM at
37 °C, except for the nystatin treatment which was in Krebs-Hepes buffer (140 mM NaCl, 4
mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 1 mM MgCl2, 5 mM HEPES, pH 7.4, 11.7 mM
glucose, 0.2% bovine serum albumin) (19). Prior to the addition of the peptides, the HeLa cells
were pretreated with the inhibitors for 30 min except in the case of brefeldin A (10 min) and
sodium azide (60 min). The internalized peptides were quantified after a 1-hour incubation of
the cells with the peptides in the presence of the inhibitors. Treatment with soluble sulfated
polysaccharide (25 µg/ml each) was conducted in serum-free α-MEM for 1 h at 37 °C. The
protein content in the cell lysate was determined by the method of Lowry (20) using a Bio-Rad
protein assay kit (Bio-Rad, Richmond, CA) with bovine γ-globulin as the standard.
Lactate dehydrogenase release assay
To assess the possible impairment of cell membranes caused by the treatment with arginine-rich
peptides, the lactate dehydrogenase (LDH) release assay (21) was conducted using a LDH
release assay kit (Wako). HeLa cells were incubated with serum-free α-MEM containing 100
µM of an arginine-rich peptide or 20 µM of mastoparan. After 3 h, the medium was collected
and mixed with substrate solution containing nitroblue tetrazolium, diaphorase, and NAD. The
mixtures were left at room temperature for 5 min, then the reaction was terminated with the stop
solution (0.5 M HCl). The absorbance at 560 nm was then measured. The LDH release activity
of the peptides was calculated as the percentage of the released LDH from the peptide-treated
cells over that treated with 0.5% (v/v) Tween 20 under the same conditions. The release from
the PBS-treated cells was taken as the control.
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RESULTS
Quantification of cellular uptake of arginine-rich peptides—In a previous report, we showed
that not only Tat-(48-60), but also many arginine-rich RNA- and DNA-binding peptides were
membrane permeable and had the ability to bring exogenous proteins into cells (14). These
results were suggestive of the presence of an ubiquitous internalization mechanism commonly
shared by these peptides. To more precisely understand the features of the internalization of
these peptides quantification of the internalized peptides was conducted. The peptides that
showed efficient translocation by the fluorescence microscopic observation were chosen as
models for the quantification, namely, HIV-1 Rev-(34-50), FHV coat-(35-49), and the (Arg)8
peptides as well as the HIV-1 Tat-(48-60) peptide (Table 1). The (Arg)16 peptide that showed the
lower membrane permeability (14) was also examined. The peptides were synthesized to have
an extra cysteine or glycyl-cysteine amide at their C-terminus for fluorescence labeling. In our
previous report, we employed the fluorescein-labeled peptide for the fluorescence microscopic
observations (14). The rhodamine-labeled peptides were used in this study for quantification
because the fluorescence intensity of fluorescein can easily be affected by the surrounding
conditions (22). After confirming that both the fluorescein-labeled and rhodamine-labeled
arginine-rich peptides were internalized into cells basically in the same manner as each other
(data not shown), the HeLa, RAW264.7 and COS-7 cells were incubated in the medium
containing the rhodamine-labeled peptides for 3 h at 37 °C, washed by PBS and solubilized in
0.5% Triton X-100 in PBS. The cellular uptake of each peptide was determined by the
fluorescence intensity of the lysate of the peptide-treated cells and corrected by the total protein
amount. Although the PBS wash was employed here to remove away non-specifically bound
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peptides on the cell surface, washing the cells with a high-salt buffer [20 mM HEPES
containing 2 M NaCl (pH 7.4)] produced little difference in the amount of peptides compared
with the case of that washed by PBS only (data not shown).
The results of the quantification are shown in Fig. 1. Although slight differences in the
cell lines were observed, the Tat-(48-60) and Rev-(34-50) showed the highest levels of
accumulation in 3 h. The accumulation of (Arg)8 was slightly less than those for the Tat-(48-60)
and Rev-(34-50). On the other hand, although the translocation efficiency of FHV coat-(35-49)
had been estimated to be comparable with the above peptides by fluorescence microscopic
observations (14), a significant difference was recognized in the quantification of the
internalized FHV coat-(35-49) from those of Tat-(48-60) and Rev-(34-50). The (Arg)16 showed
the least efficient translocation as judged from the fluorescence microscopic observations (14).
Time-course and concentration dependence on the uptake of arginine-rich peptides—Tat-(48-
60) has already been reported to enter the cells so rapidly as to reach the nucleus within 5 min
(1). To obtain information about the kinetics on the internalization of the Rev-(34-50) and
(Arg)8 peptides, the time course of the uptake of Rev-(34-50) and (Arg)8 as well as Tat-(48-60)
by HeLa cells was studied (Fig. 2). The rate of cellular uptake of Tat-(48-60) turned out to be
slightly faster than the other two peptides. The concentration of the Tat-(48-60) in the cells
reached the maximum in 2 h, and then showed a slight decrease. On the other hand, those of
Rev-(34-50) and the (Arg)8 peptides kept increasing during the observed time period. After 2 h,
the cellular concentration of the Rev-(34-50) reached almost a comparable level as that of the
Tat-(48-60).
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The dependence of the peptide concentration on the uptake was also examined. HeLa
cells were incubated for 1 h with different concentrations (0-10 µM) of Tat-(48-60), Rev-(34-
50) and (Arg)8 (Fig. 3). The amount of the internalized peptide increased as the applied peptide
concentration increased. However, the convex manner of the increase implied the presence of
saturation in the cellular concentration of the peptides. Treatment using the higher
concentrations (>20 µM) of the peptides induced cytotoxicity, and we were not able to
quantitatively ascertain whether the peptides showed maximum concentration in the cells. The
toxicity of the peptides was attributed to the rhodamine moiety, because non-labeled peptides
showed much less toxicity to the cells as described later in this report. The internalized manner,
observed in the time-course and dose dependent experiments for the Tat-(48-60), Rev-(34-50),
and (Arg)8 peptides, was almost identical to each other. This above tendency was also basically
similar with the results obtained by Polyakov (4), where the [99mTc] labeled Tat-(48-57) peptide
was used for monitoring the internalization to human leukemia Jurkat cells, although some
differences were observed in the kinetics of the uptake presumably due to the difference in the
cell lines.
Competitive inhibition of internalization of the arginine-rich peptides—As described above, the
uptake of these arginine-rich peptides seemed to be saturable. To confirm this possibility, HeLa
cells were incubated with a fluorescein-labeled peptide together with an excess amount (100
µM) of the non-labeled peptide. After the addition of the non-labeled peptide, the labeled
peptide was added to the culture medium, and the cells were incubated at 37 °C for 3 h. During
the peptide treatment, there was no detectable change in the cell morphology. After incubation,
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the cells were washed with PBS, and fixed. As a result, the internalization of the fluorescein-
labeled Tat-(48-60) and Rev-(34-50) turned out to be inhibited in the presence of an excess
amount of the corresponding non-labeled peptides (Fig. 4). A similar result was obtained in the
experiment using (Arg)8 (data not shown). Thus, it was confirmed that the internalization of
these peptides was saturable.
Next, we examined whether the internalization of a peptide could be inhibited in the
presence of other arginine-rich peptides. Although there is no common structural or sequential
characteristics except that they are all rich in arginine residues, they showed very similar
internalization characteristics. This fact raised the possibility of peptides sharing a common
pathway. Therefore, we examined whether these peptides shared a common pathway for their
internalization. The above experiments were thus conducted using the fluorescein-labeled Tat-
(48-60) or Rev-(34-50) peptides with the non-labeled other peptides.
The internalization of Tat-(48-60) was suppressed by the excess amount of Rev-(34-50)
(100 µM) to almost the same extent as in the presence of an excess amount of Tat-(48-60) itself
(Fig. 4). A similar result was obtained for Rev-(34-50) in the presence of the Tat-(48-60). An
excess amount of the (Arg)8 peptide also inhibited the internalization of the Tat-(48-60) and
Rev-(34-50) peptides (data not shown). Using RAW264.7 cells, the same results were obtained
(data not shown). These results strongly suggested that there seemed to be a common pathway
for the internalization, or a maximum intracellular concentration for these arginine-rich
peptides.
Effect of endocytosis inhibitors and metabolic inhibitors on the uptake—It has already been
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reported that the internalization of Tat-(48-60) was not influenced by the treatment of the cells
with various endocytosis inhibitors (4). To examine if this result was also applicable to the
uptake of other arginine-rich peptides, the effects of the following reagents, known as typical
endocytosis inhibitors, on the internalization of the Rev-(34-50) and (Arg)8 peptides as well as
the Tat-(48-60) were examined; the microtubule disrupting reagents; colchicine (20 µM) (23),
taxol (20 µM) (24), nocodazole (20 µM) (25); the filament disrupting reagent; cytochalasin D (5
µM) (26); the inhibitor of trans-Golgi transport; brefeldin A (10 µM) (27); the
phosphatidylinositol-3 kinase inhibitor; wortmannin (50 nM) (28); and the inhibitor of the
acidification of endocytic vesicles; chloroquine (50 µM) (29). Prior to the addition of the
peptides, the HeLa cells were incubated in serum-free medium containing each of the reagents
at 37 °C for 30 min except in the case of the brefeldin A (10 min), and then a peptide was added
to the medium. Cells were incubated at 37 °C for 1 h and the internalized peptide was quantified.
The concentration and preincubation time for these inhibitors were established by referring to
the literature (23-29). As a result, all the reagents had little or no effects on the cellular uptake of
the Rev-(34-50) and (Arg)8 peptides as in the case of the Tat-(48-60) peptide. Treatment of the
cells with these reagents at the given concentrations produced few observable morphological
differences among the cells. Treatment of the cells with sodium azide (10 mM) (30) or rotenone
(1 µM) (31), which were known to induce ATP-depletion, showed substantially no inhibitory
effect on the uptake (Table 2). Recently, Eguchi et al. reported a new approach for the
intracellular gene delivery using a lambda phage expressing the Tat segment on the phage
surface (32), where the internalization of the phage was attributed through the caveolae
mediated pathway (33). Therefore, we examined the effect of nystatin (50 µg/ml), a reagent that
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disrupts the internalization via caveolae, but it also had little effect on the uptake. These results
suggested that the typical endocytosis mechanisms would not be the main pathway but some
energy independent pathway would be involved in their internalization.
Effect of the peptide treatments on cell membrane integrity—Amphiphilic basic peptides such as
mastoparan and magainine have been known to form a helical structure in the membrane and to
assemble to form a pore to the membrane, which leads to leakage of the intracellular
components (34-36). There might be a possibility of the translocation of the Tat-(48-60), Rev-
(34-50), and (Arg)8 peptides proceeding in a similar fashion. Even without obvious pore
formation, these peptides may perturb the membrane that leads to leakage of the cellular
contents. Especially, Rev-(34-50) was suggested to form a helical structure in the membrane as
in the case of the mastoparan and magainine (14). To inquire about this possibility, we
conducted the LDH release assay (21) with peptide-treated cells to assess the effect on the
membrane permeability by the addition of the peptides. The result of LDH release assay was
shown in Fig. 5. No significant perturbation was observed by the treatment of the arginine-rich
peptides (100 µM, 3 h) which suggested that these peptides were internalized without producing
a critical membrane perturbation. On the other hand, mastoparan (20 µM) gave a significant
perturbation to the membrane. Simultaneously, the MTT assay (37) was conducted to assess the
cytotoxicity of these peptides on the HeLa cells (Fig. 6). Although Rev-(34-50) induced a slight
decrease in cell viability (~15%) at 100 µM (incubation: 24 h), Tat-(48-60) and (Arg)8 produced
substantially no obvious cytotoxic effects. A significant decrease in cell viability was not
observed when the cells were incubated with Rev-(34-50) at the lower concentration (10 µM)
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(data not shown). Considering that these peptides were not toxic to RAW264.7 cells under the
same conditions (14), these arginine-rich peptides seemed to be internalized into cells without
causing serious perturbation to the membranes.
Possible contribution of the cell surface sulfated polysaccharides to internalization—As these
arginine-rich peptides were rich in positive charges, the possible electrostatic interaction
between the arginine-rich peptides and cell-surface sulfated polysaccharides may contribute to
the internalization of the peptides. Actually, in the case of the full length Tat protein, which also
shows membrane permeability, the involvement of the cell surface heparan sulfate with its
internalization has been pointed out (16, 17). It remains unclear whether the heparan sulfate
(HS) may contribute to the translocation of Tat-(48-60) and the other arginine-rich peptides.
To confirm the feasibility of the hypothesis, fluorescence microscopic observations
were conducted. HeLa cells were preincubated in the serum-free medium containing 50 µg/ml
of HS, 0.5 U/ml of heparinase III, or 12.5 µg/ml of anti-heparan sulfate antibody for 30 min
followed by the addition of the fluorescein-labeled Rev-(34-50) (1 µM), and incubated for 30
min. As shown in Fig. 7, the fluorescence intensity from the cells was diminished by the above
treatments. Especially, a marked decrease in the nuclear localization of the peptide was
recognized. These results suggested the possible contribution of HS for the peptide
internalization. To obtain further information on this problem, we next conducted a quantitative
examination of the cellular uptake of Tat-(48-60), Rev-(34-50) and (Arg)8 in the presence of HS,
CS-A, B or C. HeLa cells were treated with 25 µg/ml of HS, CS-A, B or C for 1 h and then the
rhodamine-labeled peptide (1 µM) was added to the medium. After an additional incubation for
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1 h, the cells were lysed, and the fluorescence intensity was measured as described above.
Treatment with the sulfated polysaccharides induced a significant decrease in the cellular uptake
of Tat-(48-60), Rev-(34-50) and (Arg)8 (Fig. 8). These results further support the possibility of
the contribution of sulfated polysaccharides with the cell membrane penetration of the arginine-
rich peptides.
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DISCUSSION
In this report, we have confirmed that the HIV-1 Rev-(34-50) and (Arg)8 peptides
showed very similar translocation characteristics with HIV-1 Tat-(48-60) through quantification
and fluorescence microscopic observations of the internalized peptides. These characteristics
included the facile and dose-dependent manner of internalization. The internalization of these
peptides was saturable and competitively inhibited in the presence of other peptides. These facts
strongly suggested the possibility of these peptides sharing a common or very similar
internalization pathway. The internalization may not be explainable by typical endocytosis
mechanisms, since the major endocytosis and metabolic inhibitors were ineffective. Significant
perturbation of the cell membranes was not recognized. We have also shown that cell surface
sulfated polysaccharides could contribute to the interaction of these peptides.
However, it is still unclear how these peptides penetrate through the cell membrane
following the possible adsorption to the outside of the plasma membrane via sulfated
polysaccharides. Clathrin coated-pit mediated endocytosis (38), which is a typical energy
dependent endocytosis pathway, would not be a major route for internalization, because various
endocytosis inhibitors and metabolic inhibitors had little effect on the uptake. Recently, Eguchi
et al. reported that a lambda phage expressed the basic domain of the Tat protein on its surface
internalized into mammalian cells via the caveolae mediated pathway (32). Therefore, we
examined the possible involvement of the caveolae-mediated endocytosis with the
internalization of the arginine-rich peptides. Caveolae are small flask-shaped invaginations of
the plasma membrane and are rich in cholesterol and glycosphingolipids (33). Nystatin, which
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has been known to disrupt caveolae formation and inhibits caveolae-mediated internalization
(33), was used for the pretreatment of the cells. However, no significant effect was observed.
Liu et al. recently reported that the low-density lipoprotein receptor-related protein (LRP) was
involved in the internalization of the full length Tat protein into neural cells (16). However, this
mechanism would not be necessarily applicable to the internalization of the Tat-(48-60) peptide,
since (i) not only the basic domain (positions 48-57), but also the core domain (positions 37-48)
of the Tat protein was reported to play a crucial role in the LRP-mediated internalization (16),
and (ii) internalization of the Tat-(48-60) peptide as well as the Rev-(34-50) and (Arg)8 peptides
was only slightly affected by the sodium azide or rotenone treatment, where the LRP-mediated
pathway should be significantly suppressed. Moreover, (iii) it has been reported that there is a
certain difference in the expression of the LRP among the cells or organs (39), which should
have a certain influence on the internalization of the Tat-(48-60) peptide. However, as Tat-(48-
60) has been reported to be able to enter a variety of different cell types, this might not be the
case. Thus, we assume that the internalization mechanism of the Tat protein and the Tat-(48-60)
peptide would not be exactly the same. Actually, Liu et al. did not exclude the possibility of the
existence of energy-independent and HS dependent pathways for the internalization of the Tat
protein (16).
In this report, we have shown the possible contribution of HS for the internalization of
the arginine-rich peptides. The guanidino moiety in arginine is a strong base of pKa ~12. HS has
known to be ubiquitously expressed on animal cell surfaces. It would be possible that the strong
negatively charged HS is involved in the initial stage of the internalization pathway of these
arginine-rich peptides, or, at least, to assist these peptides to adhere to the membranes.
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Involvement of HS in the internalization of the full length Tat protein was also pointed out (16,
17). However, a difference occurs in the specificity of the sulfated polysaccharides on the
internalization of the Tat protein and the arginine-rich peptides including Tat-(48-60).
Internalization of the Tat protein was inhibited by HS, but not by the chondroitin sulfates (17),
whereas that of Tat-(48-60), Rev-(34-50), and (Arg)8 was inhibited both by the addition of HS
and the chondroitin sulfates. This fact also suggested that the internalization mechanisms of the
Tat protein and the Tat-(48-60) are not exactly the same. For the Tat protein, a certain secondary
or tertiary structure of the protein would be important, which distinguishes HS from the
chondroitin sulfates. For the internalization of the Tat-(48-60) as well as the Rev-(34-50) and
(Arg)8 peptides, an electrostatic interaction would play a crucial role, since there are few
similarities in their sequences and possible secondary structures (14).
It is not clear at this stage whether the sulfated polysaccharides are indispensable to the
membrane permeation of the arginine-rich peptides, because internalization of the Rev-(34-50)
peptide was not completely inhibited by the heparan sulfate lyase or anti-HS antibody treatment
(Fig. 7). Internalization of the Tat-(48-60) peptide to the HS-deficient cells (CHO-A745) was
preliminary reported (40). However, as the internalization itself was actually suppressed by the
heparan sulfate lyase or anti-HS antibody treatment as well as by the addition of soluble sulfated
polysaccharides, it would be plausible that the sulfated polysaccharides may contribute to
concentrating the peptides on the cell surface. Therefore, we believe that the sulfated
polysaccharides would, at least, play some part in the translocation of the basic peptides. It
would be possible that more than one mechanism is involved in the translocation. The
relationship to the adsorptive-mediated endocytosis (41, 42), which is an energy dependent
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pathway advocated for the cellular uptake of basic medicines and peptides, should be clarified.
Direct interaction of the peptides with lipid membranes could be expected as was observed in
the Antennapedia homeodomain peptide, another well-known membrane permeable basic
peptide, going through the vesicular membrane (43). The low membrane perturbable way of
internalization of the Antennapedia peptide, judged by the fluorescein leakage from the vesicles,
showed a similar tendency to the result of the LDH release assay of the Tat-(48-60), Rev-(34-
50), and (Arg)8 peptides obtained in this study.
The results presented here strongly suggested the presence of a pathway ubiquitously
lying among the internalization of basic peptides. It would be conceivable that the elucidation of
this pathway will provide a novel method of membrane traffic, which would be very important
not only for intracellular protein and drug delivery but also for the cellular metabolism and viral
infection.
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FOOTNOTES
*This work was supported by Grants-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports and Culture of Japan. The authors are grateful to Dr. H. Mukai
(Japan Tobacco Inc.) for his helpful discussions.
1The abbreviations used are: HIV, human immunodeficiency virus; FHV, flock house virus; HS,
heparan sulfate; LRP, low-density lipoprotein receptor-related protein; Fmoc, 9-
fluorenylmethyloxycarbonyl; HPLC, high performance liquid chromatography; α-MEM, alpha-
minimum essential medium; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-
buffered saline; CS, chondroitin sulfate; LDH, lactate dehydrogenase; MTT, [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; SD, standard deviation.
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FIGURE LEGENDS
Figure 1. Quantification of cellular uptake of arginine-rich peptides. Mouse macrophage
RAW264.7 (a), human cervical carcinoma HeLa (b), and simian kidney COS-7 (c) cells were
used. Cells were incubated with medium containing a rhodamine-labeled peptide (1 µM) for 3 h,
and then washed and lysed. Quantification was conducted by measuring the fluorescence
intensity of the lysate. Error bars represent the mean ± standard deviation (SD) between
triplicates.
Figure 2. Time-course of the cellular uptake of Tat-(48-60) (�), Rev-(34-50) (�), and (Arg)8
(△) by HeLa cells. Cells were incubated with fresh medium containing a rhodamine-labeled
peptide (1 µM). Quantification of the intracellular translocated peptides was conducted as
described under “Experimental Procedures.” Each point represents the mean ± SD of three
samples.
Figure 3. Concentration dependence of Tat-(48-60) (�), Rev-(34-50) (�), and (Arg)8 (△) on
the internalization into HeLa cells. Cells were incubated at 37 °C for 1 h in fresh medium
containing a given concentration of rhodamine-labeled peptides. Each point represents the mean
± SD of three samples.
Figure 4. Competitive inhibition of the internalization of fluorescein-labeled peptides in the
presence of an excess amount of non-labeled peptide. An excess of non-labeled Tat-(48-60) or
Rev-(34-50) (100 µM) was added to the culture medium, and then fluorescein-labeled peptides
(1 µM) were applied. After a 3-hour incubation, cells were washed, fixed and the intracellular
localization of the peptides was monitored by fluorescence microscopy. Pictures of the cells
treated with fluorescein-labeled Tat-(48-60) (1 µM) (A) and Rev-(34-50) (1 µM) (B) in the
absence (a), or in the presence of non-labeled Tat-(48-60) (100 µM) (b), and Rev-(34-50) (100
µM) (c) are shown, respectively.
Figure 5. Effect of the peptides on the cell membrane integrity. HeLa cells were incubated in the
serum-free medium containing 100 µM of a peptide except mastoparan (20 µM) for 3 h. The
LDH release assay was then conducted as described under “Experimental Procedures.” Error
bars represent the mean ± SD of 6-10 samples.
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25
Figure 6. Cytotoxicity of Tat-(48-60), Rev-(34-50) and (Arg)8. HeLa cells were incubated in the
medium containing 100 µM of a peptide for 24 h. The MTT assay was then conducted as
previously described (14). Error bars represent the mean ± SD of 6-7 samples.
Figure 7. Inhibition of the internalization of the fluorescein-labeled Rev-(34-50) (1 µM) by the
treatment of HeLa cells with anti-HS antibody (b), HS (c) or heparinase III (d). HeLa cells were
pretreated with anti-HS antibody (12.5 µg/ml), HS (50 µg/ml) or heparinase III (0.5 U/ml) for
30 min and then fluorescein-labeled Rev-(34-50) was added to the medium. After a 30-minute
incubation, the cells were washed, fixed and observed by fluorescence microscopy as described
above. Cells treated with PBS were used as the control (a).
Figure 8. Quantification of the cellular uptake of Tat-(48-60), Rev-(34-50) and (Arg)8 by HeLa
cells in the presence of sulfated polysaccharide. Cells were treated with HS (a), CS-A (b), CS-B
(c) or CS-C (d) (25 µg/ml each) in serum-free medium for 30 min, and subsequently, a
rhodamine-labeled peptide (1 µM) was added to the culture medium. After a 1-hour incubation,
the cells were washed, lysed and the fluorescence intensity of the lysate was measured as
described above. Error bars represent the mean ± SD of three samples.
Table 1. Primary structures of the membrane-permeable arginine-rich peptides used in this study.
The C-terminal cysteine was fluorescein- or rhodamine-labeled for monitoring or quantification
of the peptide internalization, respectively.
Table 2. Effect of various reagents on the cellular uptake of Tat-(48-60), Rev-(34-50) and (Arg)8.
The cells were treated with these reagents at the given concentrations, and then a peptide (1 µM)
was added to the medium. The cells were incubated for another 1 h and subjected to
quantification. The data were the means of three samples, expressed by the % of the internalized
peptide into cells without treatment of the inhibitors.
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peptides sequences
HIV-1 Tat-(48-60)
FHV coat-(35-49)
NH2-GRKKRRQRRRPPQ-C-CONH2
NH2-RRRRNRTRRNRRRVR-GC-CONH2
NH2-RRRRRRRR-GC-CONH2(Arg)8
NH2-RRRRRRRRRRRRRRRR-GC-CONH2(Arg)16
Table 1. Primary structures of the membrane-permeable arginine-rich peptides used in
this study. The C-terminal cysteine was fluorescein- or rhodamine-labeled for monitoring
or quantification of the peptide internalization, respectively.
HIV-1 Rev-(34-50) NH2-TRQARRNRRRRWRERQR-GC-CONH2
26
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wortmannin (50 nM)
cytochalasin D (5 µM)
colchicine (20 µM)
Tat Rev (Arg)8reagents
nocodazole (20 µM)
taxol (20 µM)
brefeldin A (10 µM)
chloroquine (50 µM)
92.9 % 93.9 % 95.2 %
102.0 % 102.2 % 100.8 %
95.5 % 93.9 % 90.7 %
93.5 % 88.3 % 108.7 %
94.6 % 94.3 % 95.6 %
106.1 % 107.6 % 104.2 %
99.7 % 99.9 % 92.8 %
a) endocytosis inhibitors
b) metabolic inhibitors
sodium azide (10mM)
rotenone (1 µM)
Tat Revreagents
87.0 % 96.9 % 101.9 %
92.0 % 99.5 %102.8 %
c) caveolae formation inhibitor
nystatin (50 µg/ml)
Tat Revreagent
109.0 % 92.5 %97.6 %
(Arg)8
(Arg)8
Table 2. Effect of various reagents on the cellular uptake of Tat-(48-60), Rev-(34-50) and (Arg)8.
The cells were treated with these reagents at the given concentrations, and then a peptide (1 µM) was
added to the medium. The cells were incubated for another 1 h and subjected to quantification. The
data were the means of three samples, expressed by the % of the internalized peptide into cells
without treatment of the inhibitors.
27
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Tat Rev FHV (Arg)8 (Arg)16
up
take
(p
mo
l/m
g p
rote
in)
(a) RAW264.7 (b) HeLa (c) COS-7
Tat Rev FHV (Arg)8 (Arg)16 Tat Rev FHV (Arg)8 (Arg)16
800
700
600
500
400
300
200
100
0
Figure 1. Quantification of cellular uptake of arginine-rich peptides. Mouse macrophage
RAW264.7 (a), human cervical carcinoma HeLa (b), and simian kidney COS-7 (c) cells were
used. Cells were incubated with medium containing a rhodamine-labeled peptide (1 µM) for 3
h, and then washed and lysed. Quantification was conducted by measuring the fluorescence
intensity of the lysate. Error bars represent the mean ± standard deviation (SD) between
triplicates.
28
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500
400
300
200
100
030 60 90 120 150 1800
incubation time (min)
up
take
(p
mo
l/m
g p
rote
in)
Figure 2. Time-course of the cellular uptake of Tat-(48-60) (l), Rev-(34-50) (s), and (Arg)8 ( )
by HeLa cells. Cells were incubated with fresh medium containing a rhodamine-labeled peptide (1
µM). Quantification of the intracellular translocated peptides was conducted as described under “
Experimental Procedures.” Each point represents the mean ± SD of three samples.
29
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0 2 4 6 8 10
250
500
750
1000
1250
1500
0
concentration (µM)
up
take
(p
mo
l/m
g p
rote
in)
Figure 3. Concentration dependence of Tat-(48-60) (l), Rev-(34-50) (s), and (Arg)8 ( ) on
the internalization into HeLa cells. Cells were incubated at 37 °C for 1 h in fresh medium
containing a given concentration of rhodamine-labeled peptides. Each point represents the
mean ± SD of three samples.
30
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(a) control
fluorescein-
labeled
Tat-(48-60)
(b) Tat-(48-60) (100 µM)
(1 µM)
(1 µM)
(c) Rev-(34-50) (100 µM)
A
fluorescein-
labeled
Rev-(34-50)
B
Figure 4. Competitive inhibition of the internalization of fluorescein-labeled peptides in the
presence of an excess amount of non-labeled peptide. An excess of non-labeled Tat-(48-60)
or Rev-(34-50) (100 µM) was added to the culture medium, and then fluorescein-labeled
peptides (1 µM) were applied. After a 3-hour incubation, cells were washed, fixed and the
intracellular localization of the peptides was monitored by fluorescence microscopy. Pictures
of the cells treated with fluorescein-labeled Tat-(48-60) (1 µM) (A) and Rev-(34-50) (1 µM)
(B) in the absence (a), or in the presence of non-labeled Tat-(48-60) (100 µM) (b), and Rev-(3
4-50) (100 µM) (c) are shown, respectively.
31
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LD
H r
ele
ase
(%
)100
80
60
40
20
0
mastoparan Tat Rev (Arg)8
-10
Figure 5. Effect of the peptides on the cell membrane integrity. HeLa cells were incubated in
the serum-free medium containing 100 µM of a peptide except mastoparan (20 µM) for 3 h.
The LDH release assay was then conducted as described under “Experimental Procedures.”
Error bars represent the mean ± SD of 6-10 samples.
32
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100
80
60
40
20
0
via
bili
ty (
%)
CTL Tat Rev (Arg)8
Figure 6. Cytotoxicity of Tat-(48-60), Rev-(34-50) and (Arg)8. HeLa cells were incubated
in the medium containing 100 µM of a peptide for 24 h. The MTT assay was then
conducted as previously described (14). Error bars represent the mean ± SD of 6-7 samples.
33
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Figure 7. Inhibition of the internalization of the fluorescein-labeled Rev-(34-50) (1 µM) by
the treatment of HeLa cells with anti-HS antibody (b), HS (c) or heparinase III (d). HeLa
cells were pretreated with anti-HS antibody (12.5 µg/ml), HS (50 µg/ml) or heparinase III
(0.5 U/ml) for 30 min and then fluorescein-labeled Rev-(34-50) was added to the medium.
After a 30-minute incubation, the cells were washed, fixed and observed by fluorescence
microscopy as described above. Cells treated with PBS were used as the control (a).
34
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0
100
200
300
400
500
600
700
(a) HS (b) CS-A
(c) CS-B (d) CS-C
Tat Rev (Arg)8Tat Rev (Arg)8
Tat Rev (Arg)8Tat Rev (Arg)8
up
take
(p
mo
l/m
g p
rote
in)
0
100
200
300
400
500
600
700
up
take
(p
mo
l/m
g p
rote
in)
Figure 8. Quantification of the cellular uptake of Tat-(48-60), Rev-(34-50) and (Arg)8 by
HeLa cells in the presence of sulfated polysaccharide. Cells were treated with HS (a), CS-A
(b), CS-B (c) or CS-C (d) (25 µg/ml each) in serum-free medium for 30 min, and
subsequently, a rhodamine-labeled peptide (1 µM) was added to the culture medium. After a 1
-hour incubation, the cells were washed, lysed and the fluorescence intensity of the lysate was
measured as described above. Error bars represent the mean ± SD of three samples.
35
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SugiuraTomoki Suzuki, Shiroh Futaki, Miki Niwa, Seigo Tanaka, Kunihiro Ueda and Yukio
peptidesPossible existence of common internalization mechanisms among arginine-rich
published online November 15, 2001J. Biol. Chem.
10.1074/jbc.M110017200Access the most updated version of this article at doi:
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