From the Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
Received for publication, August 27, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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Cationic antimicrobial peptides play
important roles in innate immunity. Compared with extensive studies on
peptide-bacteria interactions, little is known about peptide-human cell
interactions. Using human cervical carcinoma HeLa and fibroblastic TM12
cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and
energy dependence of translocation suggested that the three peptides
cross cell membranes through different mechanisms. The magainin peptide
was internalized within a time scale of tens of minutes. The
cooperative concentration dependence of uptake suggested that the
peptide forms a pore as an intermediate similar to the observations in
model membranes. Furthermore, the translocation was coupled with
cytotoxicity, which was larger for tumor HeLa cells. In contrast, the
buforin peptide translocated within 10 min by a
temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the
highest concentration investigated (100 µM). The
uptake of the Tat peptide was proportional to the peptide
concentration, and the concentration dependence was lost upon ATP
depletion. The peptide exhibited a moderate cytotoxicity at higher
concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa
green fluorescent protein, also entered cells, suggesting a potency of
the peptide as a vector for macromolecular delivery into cells.
However, the mechanism appeared to be different from that of the parent peptide.
Cationic antimicrobial peptides play important roles in
innate immunity (1-4). Broad antimicrobial spectra, highly selective toxicity, as well as difficult resistance development make these compounds promising candidates as novel antibiotics for clinical use.
Intensive investigations on peptide-bacteria interactions have shown
that some peptides kill bacteria mainly by permeabilizing the
membranes, whereas others target intracellular compounds, such as
nucleic acids, although recent studies have suggested that many
peptides possess both modes of action (2, 4, 5).
Magainin 2 (Table I) isolated from the skin of the African clawed
frog Xenopus laevis is a representative
membrane-acting peptide composed of 23 amino acid residues (6-8).
Addition of the peptide to bacterial cells immediately induces the
permeabilization of both outer and inner membranes, leading to cell
death (9, 10). A fluorescent magainin peptide mainly associates with
bacterial membranes and does not enter cells (10), although native
magainin 2 was recently reported to enter cells (11). An example of the peptides targeting intracellular substances is buforin 2, which was
discovered in the stomach of the Asian toad Bufo bufo
gargarizans (12). A fluorescent buforin 2 rapidly enters bacterial
cells without perturbing the barrier properties of the membranes,
binding to DNA and RNA (10).
On the other hand, interactions of cationic antimicrobial peptides with
human cells are not well characterized. The information on cell
penetration available is limited to dermaseptins (13-15), PR-39 (16),
and protegrin 1 (17), although many investigators examined hemolytic
activity (9, 18) and cytotoxicity (19-22). In contrast, short Arg-rich
cationic peptides have recently been shown to enter mammalian cells by
an energy (ATP)-independent, non-endocytotic pathway and have been
utilized as vectors for delivering membrane-impermeable drugs,
oligonucleotides, and proteins into cells (23). Model membrane studies
revealed that the membrane-acting magainin peptide also translocates
across lipid bilayers (24, 25), although less effectively than buforin
2 (18). Therefore, cationic antimicrobial peptides, including those of
the membrane-permeabilizing class, can penetrate human cells. However,
although this issue has rarely been investigated, it is important for
the development of novel peptides for therapeutic use as well as for
understanding the roles of antimicrobial peptides in innate immunity.
In this study, we investigated the translocation of fluorescent-labeled
analogues of magainin 2 and buforin 2 across human cell membranes in
comparison with the Tat-(47-57) peptide (Table I), a representative
Arg-rich cell-penetrating peptide. Both tumor (human cervical carcinoma
HeLa) and normal (human fibroblastic TM12, Ref. 26) cells were used
because magainin peptides are known to exhibit selective toxicity for
tumor cells (19-21, 27). The dose, time, temperature, and energy
dependence of translocation suggested that the three peptides crossed
cell membranes through different mechanisms. Furthermore, the buforin
peptide, covalently attached to the 28-kDa green fluorescent protein
also entered cells, suggesting a potency of the peptide as a vector for
macromolecular delivery into cells.
Materials--
The reagents for peptide synthesis were purchased
from Applied Biosystems (Foster City, CA). All fluorescent probes for
peptide labeling were obtained from Molecular Probes (Eugene, OR).
Dulbecco's modified Eagle's medium (DMEM) was supplied by nacalai
tesque (Kyoto, Japan). Bis(sulfosuccinimidyl)suberate
(BS3)1 linker and
MTT were purchased from Pierce and Sigma, respectively. GFP was
supplied by Upstate Biotechnology (Lake Placid, NY). All other
chemicals from Wako (Tokyo, Japan) were of special grade.
Peptide Synthesis--
The peptide was synthesized by a standard
fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid phase method on an
Applied Biosystems Model 433A peptide synthesizer, as previously
described (28). Fluorescent labeling of the peptides was performed as
follows. After cleavage from the resin, the Cys residue of the magainin peptide or the buforin analogue dissolved in 5 mM phosphate
buffer (pH 7.4) was conjugated with thiol-reactive Texas Red(R)
C2 maleimide in acetonitrile/methanol (1:1, v/v). The N
terminus of the Tat peptide on the resin was labeled with Texas
Red(R)-X succinimidyl ester in
N,N-dimethylformamide and isopropylethylamine,
washed with N,N-dimethylformamide and
acetonitrile, and then cleaved from the resin. The purity of the
synthesized peptides was determined by analytical reversed phase-high
performance liquid chromatography (RP-HPLC) and ion spray mass spectroscopy.
Cell Culture--
HeLa and TM12 cells were cultured in DMEM with
10% fetal bovine serum (FBS). The cells were maintained under 5%
CO2 at 37 °C.
Cellular Uptake of Peptide--
The cells (1 × 104/cm2) were plated on 24-well microplates (2 cm2/well), and then incubated overnight. After removal of
the medium, the cells were exposed to peptide solutions (200 µl)
freshly dissolved in DMEM with 10% FBS for various periods at
37 °C, unless otherwise indicated. The peptide solutions were then
aspirated, and the cells were washed three times with
phosphate-buffered saline (PBS) of pH 7.4 and lysed with 0.5 ml of 10%
SDS in PBS. The protein content of the cells was determined by the
Bio-Rad DC protein assay kit (Hercules, CA). The internalized Texas
Red-labeled peptides were fluorometrically quantified with a Shimadzu
RF-5300 spectrofluorometer at an excitation wavelength of 583 nm and an
emission wavelength of 610 nm.
Effects of Low Temperature or Sodium Azide--
The assays were
performed as described above with the exception that the cells were
preincubated for 30 min either at 4 °C or in the presence of 0.5%
(w/v) sodium azide in DMEM with 10% FBS at 37 °C. The subsequently
added peptide solution was cooled at 4 °C or contained 0.5% (w/v)
sodium azide.
Confocal Laser Microscopy--
The cells (1 × 104/cm2) were plated on 8-well chamber slides
(1 cm2/well), incubated overnight, and then treated with
peptide solutions as described above. For nuclear staining, 5 µM SYTO16 (Molecular Probes) was added to the peptide
solutions. After washing three times with PBS, the cells were fixed
with 4% (w/v) paraformaldehyde in PBS for 5 min at room temperature,
washed again with PBS, and mounted in a glycerol/PBS (9:1, v/v)
solution containing 0.1% p-phenylendiamine dihydrochloride.
Microscopic examination was performed on a Bio-Rad MRC1024 confocal
laser microscope.
Cytotoxicity--
The cells (5 × 103
cells/well) were cultured in 96-well microplates and incubated
overnight in DMEM containing 10% FBS. After removal of the medium, the
cells were incubated with 0.1 ml of peptide dissolved in the medium for
24 h at 37 °C. Ten microliters of a 5 mg/ml MTT solution in PBS
was then added, and the cells were further incubated for 5 h.
Thereafter, the precipitated MTT was dissolved overnight in 0.1 ml of
20% (w/v) SDS in 0.01 M HCl. The percent cell viability
was determined by 100 × (OD595 Buforin-GFP Conjugate--
The N terminus of the buforin peptide
dissolved in sodium bicarbonate buffer (pH 8.3) was labeled with
Rhodamine Red-X succinimidyl ester in dimethylsulfoxide. Minor
byproducts were removed by RP-HPLC. The labeled peptide (65 µM) and GFP (24 µM) were mixed in PBS (pH
7.4). 30-fold excess over the protein of the amino group-reactive linker BS3 freshly dissolved in sodium citrate buffer (pH
5.0) at a concentration of 10 mg/ml was added to the mixed solution.
After 30 min, the reaction was stopped with excess 1 M
Tris. The aggregates were precipitated by centrifugation, and then the
unreacted reagents in the supernatant were removed from the buforin-GFP
conjugate by Millipore Micron centrifugal filter devices (Bedford, MA). The average number of attached buforin peptide molecules per GFP was
1.77, as estimated by UV-visible absorption.
Peptide Design--
The magainin 2 derivative (MG2d, Table
I) was designed as follows. The
translocation occurs upon the disintegration of a pore (24). The
translocation efficiency is known to be enhanced by increasing the
peptide's positive charge, which destabilizes the intermediate pore
(29). Therefore, the Glu19 residue of magainin 2 was
replaced with Gln, and the C terminus was amidated. This modification
increased the charge by +2. Texas Red, which is brighter and has a
lower background than other commonly used red fluorescent dyes and is
suited to laser excitation, was linked to the Cys residue introduced at
the 23rd position. The F12W substitution was for easy quantitation of
the unlabeled peptide by optical density at 280 nm. The Trp
introduction to the buforin 2 analogue BF2d was for the same reason.
The C-terminal extension GGC was added to link various molecules via
the SH group in future studies. In this study, Texas Red was covalently
attached to the peptide for laser microscopy. The Tat-(47-57) peptide
includes the sequence responsible for the cellular uptake of the human immunodeficiency virus-1 Tat protein consisting of the polycationic region 49-57 (30). The N terminus of the peptide was also labeled with
this dye. The labeling position had no effect on peptide-cell interactions: We also synthesized a BF2d analogue with the dye at the N
terminus. The concentration and time dependence of peptide uptake were
almost identical to those of BF2d (data not shown).
Intracellular Distribution--
The fluorescent
peptides (5 µM) were incubated with HeLa or TM12 cells
for 30 min at 37 or 4 °C, and the intracellular distributions of the
peptides were examined by confocal laser microscopy. At 37 °C, MG2d
translocated across the cell membranes of both cells (Fig.
1, A and D) and was
almost uniformly distributed inside the cells except for the nuclei in
which the peptide was less concentrated. This could be more clearly
seen by co-labeling with the nuclear-staining dye SYTO16 (Fig.
1B). Similar results were also obtained at 4 °C (Fig.
1C). In contrast to the magainin analogue, the intranuclear
concentrations of BF2d (Fig. 1E) and Tat-(47-57) (Fig.
1H) were similar to those in the cytosol. The co-labeling with SYTO16 clearly indicated that BF2d had entered the nuclei (Fig.
1F). The uptake of these peptides was also not significantly temperature-dependent (Fig. 1, G and
I). The treatment of MG2d roughened the cell surfaces
compared with the other two peptides.
To prove that most peptide molecules were not surface-bound but
internalized, we performed the optical sectioning of HeLa cells treated
with MG2d as in Fig. 1A. The focus plane was changed from
bottom to top in the vertical axis range of 32.8 µm at an interval of
1.64 µm. Representative photographs are shown in Fig. 2. Texas Red fluorescence was only
observed in the central region of the cell.
Concentration Dependence--
In addition to fluorescence
microscopy, quantitative data on the cellular uptake of the peptides
are useful for the elucidation of translocation mechanisms. Fluorescent
flow cytometry has often been utilized for this purpose (30). An
alternative, more quantitative method was used in this study. The
amount of peptide taken up was fluorometrically determined after cell
solubilization. At the same time, the amount of cellular proteins was
quantitated as a measure of the number of cells. The amount of peptide
uptake per mg of cellular protein during a 30-min incubation at
37 °C is plotted as a function of peptide concentration for HeLa and TM12 cells in Fig. 3, A and
B, respectively. Although these values include the amount of
peptide bound to the cell surface, it is clear from Figs. 1 and 2 that
most peptide molecules were internalized.
The uptake of MG2d into both cells was apparently cooperative with
respect to peptide concentration (Fig. 3, circles). The double logarithmic plots gave slopes of 1.6 (r = 0.982)
and 1.4 (r = 0.995) for HeLa and TM12 cells,
respectively (plots not shown). In contrast, the uptake of BF2d was
much less concentration-dependent (Fig. 3, squares).
The Tat-(47-57) peptide was incorporated into the cells almost
linearly to the peptide concentration of at least 15 µM
(Fig. 3, triangles).
Effects of Low Temperature and Metabolic
Inhibitor--
Endocytotic processes are known to be inhibited at
lower temperatures or by ATP depletion (31-33). Open bars
in Fig. 4A show the peptide
uptake by HeLa cells at 4 °C relative to that at 37 °C
(control). The uptake of MG2d was reduced by 40% whereas
those of BF2d and Tat-(47-57) were insensitive to temperature.
Shaded bars in Fig. 4A show the effects of sodium
azide, which blocks ATP synthesis by inhibiting cytochrome oxidase, on
peptide uptake. The internalization of MG2d was slightly (~20%)
reduced by the metabolic inhibitor while that of BF2d was not affected
by the treatment. In contrast, the azide treatment largely abolished the concentration dependence of Tat uptake (Fig. 4B). Such a
phenomenon was not observed for BF2d (data not shown).
Time Dependence--
Fig.
5, A and B show the
time dependence of peptide uptake by HeLa and TM12 cells, respectively.
For MG2d (circles), the uptake kinetics was biphasic. A
5-min incubation was sufficient for the half-maximal uptake. A longer
incubation gradually increased uptake, which was saturated at 60-120
min. The time course of BF2d internalization was completely different
(squares). The uptake was rapid and reached the maximum at
10 min, and then gradually decreased. In contrast to these
antimicrobial peptides, the Tat-(47-57) peptide was continuously taken
up at least up to 120 min (triangles).
Cytotoxicity--
The cytotoxicities of the peptides without Texas
Red labeling were evaluated by the standard MTT assay, which
demonstrates active energization of cells and is conventionally used as
a measure of cell viability. The percent viability is plotted as a
function of the peptide concentration in Fig.
6. In the presence of MG2d, the cell
viability was abruptly decreased beyond 10 and 20 µM for
HeLa (open circles) and TM12 cells (closed
circles), respectively. In contrast, BF2d was practically
non-toxic at least up to 100 µM for both cells
(squares). Upon Tat-(47-57) treatment
(triangles), the cell viability was decreased as a function
of peptide concentration over the range of 0.1 to 100%.
Buforin-Protein Conjugate--
The absence of any significant
cytotoxicity suggests that the buforin peptide is a promising candidate
as a vector for delivering macromolecules into cells. To examine this
possibility, BF2d was conjugated with GFP (28 kDa) by use of the
bifunctional cross-linker BS3. Fig.
7A shows that GFP alone did
not enter HeLa cells. In contrast, the conjugate was internalized into
the cells, as confirmed by both GFP (protein part) and Texas Red
(peptide part) fluorescence (Fig. 7B). The conjugate was
uniformly distributed within the cells and reached the nuclei,
similarly to the vector peptide (Fig. 1E). The uptake
efficiency was, however, lower than that of BF2d alone (Fig.
7D) and diminished at low temperature (Fig. 7E)
or in the presence of sodium azide (Fig. 7F).
Magainin Analogue--
Interactions of magainins with lipid
bilayers are well characterized (for review, see Refs. 7 and 8). The
peptides preferentially bind to negatively charged phospholipids,
forming amphipathic helices (28). Their affinities for mammalian cells,
on the surface of which anionic phospholipids are not exposed, have
been considered to be low, although weak hydrophobic interaction drives
peptide binding (34). The amounts of MG2d taken up by both cells were larger than those of BF2d and Tat-(47-57) (Fig. 3), despite having the
smallest positive charge. The hydrophobicity of MG2d, which has several
hydrophobic residues (2Ile, 2Phe, Trp, Leu, Val), was the largest among
the three peptides (Table I).
After membrane binding, several magainin helices form a toroidal pore
(diameter 2-3 nm) with surrounding lipid molecules in artificial
membranes (35). Upon pore disintegration, a fraction of the peptide
molecules translocates across the membrane (24). This is reflected by a
cooperativity in peptide binding, membrane permeabilization (28), and
cytotoxicity (22). The cooperativities observed in cellular uptake of
MG2d (Fig. 3) and cytotoxicity (Fig. 6) suggest that the magainin
derivative also forms a pore in cell membranes and that pore formation
and subsequent cell entry are closely related to cell death. One
possibility is that membrane permeabilization leads to cell death by
dissipating membrane potential and leakage of intracellular molecules.
Another possibility is that the peptide damages mitochondria (36),
resulting in apoptosis. The surface-roughening effect of MG2d (Fig. 1)
also suggests membrane activity of this peptide.
In studies using large unilamellar vesicles of 100 nm in diameter, the
pore formation-translocation process is biphasic and reaches
pseudo-equilibrium within ~10 min (24). In cells that are much larger
and have various organelles and macromolecules that the magainin
peptide can interact with, the internalization takes much longer,
consistent with the kinetic uptake data (Fig. 5).
The uptake of MG2d was partially inhibited at low temperature (Fig.
4A). This observation also supports translocation via the
transient formation of a toroidal pore. At low temperature, where
membranes are less fluid, the formation of the peptide-lipid supramolecular complex pore that requires membrane bending will be
suppressed. The entry of a synthetic cationic amphipathic helix composed of 18 amino acid residues was also partially inhibited at low
temperature (37), although this observation was attributed to the
presence of multiple cell permeation mechanisms. Sai et al.
(32) reported that adsorptive-mediated endocytosis of a small basic
peptide was partially inhibited at low temperature (32). However, in
this mechanism, the peptide uptake was saturated at higher peptide
concentrations. The opposite effect was observed for MG2d (Fig. 3).
Furthermore, the peptide internalized by adsorptive-mediated endocytosis showed granular distribution and was completely excluded from the nuclei. Neither was observed for MG2d (Figs. 1 and 2). Our
cytotoxicity data (Fig. 6) show that MG2d preferentially kills transformed HeLa cells over non-tumor TM12 cells, consistent with previous studies (19-21, 27).
Buforin Analogue--
Buforin 2 effectively crosses bacterial cell
membranes (10), although the mechanism has not yet been elucidated. The
observation that peptides also translocate across model lipid bilayers
without perturbing lipid organization (18) indicates that the membrane permeation occurs by a passive mechanism. Indeed, the cell entry of
BF2d was neither affected by low temperature nor the metabolic inhibitor (Fig. 4). The amount of peptide entered the cells was reduced
after 10 min, suggesting the presence of an efflux mechanism (Fig. 5).
Similar behaviors have been reported for a rhodamine-labeled Tat-(48-60) peptide (38) and a 99mTc-chelated Tat
peptide (39).
The buforin derivative effectively reached the nuclei (Fig.
1F), in keeping with a higher affinity of buforin 2 with RNA
and DNA compared with magainin 2 (10). Buforin 2 has been considered to
kill bacteria by binding to nucleic acids, therefore inhibiting protein
synthesis (10). However, BF2d did not show any significant toxicities
against both HeLa and TM12 cells at least up to 100 µM
(Fig. 6), probably because the amounts of the internalized peptide were
the lowest among the peptides investigated (Fig. 3) and because
of the presence of an efflux mechanism (Fig. 5).
The absence of cytotoxicity suggests that BF2d is a promising candidate
as a vector for intracellular delivery of membrane-impermeable cargo
molecules. A number of Arg-rich peptides have been developed for this
purpose (for review, see Ref. 23). Our BF2d-GFP conjugate could enter
cells, although the efficiency was much lower than that of the vector
peptide BF2d (Fig. 7). The observations that the cellular uptake of the
conjugate was diminished at low temperature or in the presence of
sodium azide suggest that the conjugate and the vector permeate the
cell membranes by different mechanisms.
Tat-(47-57)--
Various short Tat peptides have been reported to
permeate the cell membranes without perturbing membrane integrity and
to reach the nuclei (30, 38-40). These peptides including other Arg-rich peptides were suggested to share common non-endocytotic internalization pathway(s) (38). However, the effects of temperature and metabolic inhibitors and the time and concentration dependence of
cellular uptake appear to depend on the peptide sequences and cell
lines. Low temperature did not affect the uptake of Tat-(48-60) (40)
or Tat-(47-57) (Fig. 4) by HeLa cells. In contrast, the kinetics of
99mTc-chelated Tat peptide internalization into Jurkat
cells was largely changed by a lowering of the temperature (39). The
uptake of Tat-(48-60) (38) and 99mTc-chelated Tat (39) by
HeLa and Jurkat cells reached a maximum at 60-90 min and 5 min,
respectively, whereas that of Tat-(47-57) by HeLa cells continued to
increase at least up to 120 min (Fig. 5A). Our Tat-(47-57)
peptide was internalized proportionally to a peptide concentration of
up to 15 µM (Fig. 3A). In contrast, the uptake
of Tat-(48-60) was saturated even below 10 µM (38). Sodium azide was reported to have no effect on the uptake of
Tat-(48-60) (38). However, in our case, the concentration dependence
of uptake was completely lost by this treatment (Fig. 4B),
suggesting some involvement of energy-dependent processes.
At lower peptide concentrations, the presence of sodium azide induced a
slight enhancement in uptake. The reason for this is not clear at
present. ATP depletion can cause various changes in cell membranes,
including the inhibition of endocytosis-mediated recycling of cell
surface molecules (33), the blockage of transbilayer movement of
phospholipids (41), and the dissipation of transmembrane potential
(42). The partial reduction (20%) of MG2d uptake by the metabolic
inhibitor (Fig. 4A) may be related to these alterations in
the cell surface. Alternatively, several translocation pathways
including those that are energy-dependent may be present,
as suggested for a synthetic cationic amphipathic helix (37).
We investigated the cellular uptake of the analogues of the two
representative antimicrobial peptides, membrane-acting magainin 2 and
nuclear targeting buforin 2, in comparison with the Tat-(47-57) peptide. Together with previous reports on various cell-penetrating peptides, our data show the existence of at least three mechanisms for
cell entry. 1) The magainin-class peptide crosses the cell membranes by
the transient pore formation-translocation mechanism that can be
reproduced in artificial lipid bilayer systems (7, 8). The process is
moderately temperature-sensitive. 2) The buforin class peptide
translocates into the cells without perturbing the cell membranes by an
unknown mechanism. The translocation phenomenon is also observable in
artificial systems (18). The process is not influenced by temperature
or a metabolic inhibitor, and the cytotoxicity is minimal. 3) The
Tat-class peptide enters the cells by an unknown mechanism that many
Arg-rich peptides may share (38). The temperature and energy dependence
vary among the peptide sequences and the cell lines used. The peptides
show moderate cytotoxicities at higher concentrations (40). The
membrane permeation cannot be reproduced in liposomal
systems.2 It is not clear
whether the Antennapedia peptides (penetratins) (43) belong to this
category or not because conflicting results were reported on the
translocation across lipid bilayers (44, 45). It should be noted that
the attachment of a cargo molecule may change the translocation
mechanism, as observed for BF2d-GFP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
OD655) for peptide-treated cells/(OD595
OD655) for
untreated cells. OD595 and OD655 denote
absorbances at 595 and 655 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
Amino acid sequences of the peptides used in this study
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Fig. 1.
Intracellular distribution of
fluorescent-labeled cationic peptides as examined by confocal laser
microscopy. The MG2d, BF2d, and Tat-(47-57) peptides (5 µM) labeled by Texas Red (Table I) were incubated with
HeLa cells or TM12 cells for 30 min at 37 °C or 4 °C. The cells
were then washed and fixed, and the intracellular localization of the
peptides was examined by a confocal laser microscope.
Red shows fluorescence of Texas Red. In B and
F, fluorescence (green) of the nuclear-staining
dye SYTO16 was superimposed. The coexistence of SYTO16 and Texas Red
can be seen in yellow.
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Fig. 2.
Intracellular distribution of MG2d as
examined by optical sectioning. A HeLa cell treated with 5 µM MG2d for 30 min at 37 °C was subjected to optical
sectioning using a confocal laser microscope as described in the legend
to Fig. 1. The focus plane was changed from bottom to
top in the vertical axis range of 32.8 µm at an interval
of 1.64 µm. Representative photographs are shown.
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Fig. 3.
Concentration dependence of peptide
uptake. The peptides at various concentrations were incubated with
HeLa (A) or TM12 (B) cells for 30 min at
37 °C. The cells after washing were solubilized with 10% (w/v)
SDS-containing PBS. The amount of uptaken peptide was fluorometrically
determined and expressed as nmol of peptide per mg of cellular protein
(average ± S.D., n = 3). Peptides:
circles, MG2d; squares, BF2d;
triangles, Tat-(47-57).
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Fig. 4.
Effects of low temperature and sodium azide
on peptide uptake. The peptides (5 µM) were
incubated with HeLa cells for 30 min at 37 or 4 °C. The amount of
peptide uptake was fluorometrically determined after washing and
solubilization. Open bars show peptide uptake at 4 °C
relative to that at 37 °C. To examine the effects of sodium azide,
HeLa cells were preincubated with 0.5% (w/v) NaN3, and the
uptake experiments were performed in the presence or the absence of the
metabolic inhibitor for 30 min at 37 °C. The peptide concentration
was 5 µM. Shaded bars show peptide uptake with
NaN3 relative to that without NaN3. For
Tat-(47-57), similar experiments were carried out at various peptide
concentrations. Open and closed triangles show
peptide uptake without and with NaN3, respectively. Each
datum expresses average ± S.D. (n = 3).
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Fig. 5.
Time course of peptide uptake. The
peptides (5 µM) were incubated with HeLa (A)
or TM12 (B) cells for various periods at 37 °C. The
amount of peptide uptake was fluorometrically determined after washing
and solubilization (average ± S.D., n = 3).
Peptides: circles, MG2d; squares, BF2d;
triangles, Tat-(47-57).
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Fig. 6.
Cytotoxicity of unlabeled peptides. HeLa
cells (open symbols) or TM12 cells (closed
symbols) were incubated with various concentrations of peptide
without Texas Red labeling for 24 h at 37 °C. The cell
viability was determined by the standard MTT assay (average ± S.D., n = 3). Peptides: circles, MG2d;
squares, BF2d; triangles, Tat-(47-57).
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Fig. 7.
Intracellular distribution of the GFP-BF2d
conjugate as examined by confocal laser microscopy. The conjugate,
GFP alone, BF2d alone were incubated with HeLa cells for 30 min at
37 °C or 4 °C without or with 0.5% (w/v) NaN3. The
cells were then washed and fixed, and the intracellular localization of
each component was examined by use of GFP and Texas Red fluorescence as
markers of the protein and the peptide vector.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
CONCLUSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by the Mochida Memorial Foundation for Medical and Pharmaceutical Research, NOVARTIS Foundation (Japan) for the Promotion of Science, the Inamori Foundation, the Shimizu Foundation for the Promotion of Immunology Research, the Japan Securities Scholarship Foundation, and Grant-in-aids for Scientific Research (14572091, 13024244, and 14017048) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Graduate School of
Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel.:
81-75-753-4574; Fax: 81-75-761-2698; E-mail:
katsumim@pharm.kyoto-u.ac.jp.
Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M208762200
2 K. Matsuzaki and A. Chikushi, unpublished work.
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ABBREVIATIONS |
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The abbreviations used are: BS3, bis(sulfosuccinimidyl)suberate; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Fmoc, fluoren-9-ylmethoxycarbonyl; GFP, green fluorescent protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-buffered saline.
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