1 Title: Possible existence of common internalization mechanisms

1

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.

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1 Title: Possible existence of common internalization mechanisms