Tài liệu Báo cáo khoa học: Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat docx

Different mechanisms for cellular internalization of the HIV-1
Tat-derived cell penetrating peptide and recombinant proteins
fused to Tat
Michelle Silhol
1
, Mudit Tyagi
2
, Mauro Giacca
2
, Bernard Lebleu
1
and Eric Vive
Á
s
1
1
Institut de Ge
Â
ne
Â
tique Mole
Â
culaire de Montpellier, CNRS UMR 5124, BP5051, Montpellier, France;
2
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
Translocation through the plasma membrane is a major
limiting step for the cellular delivery of macromolecules.
A promising strategy to overcome this problem consists in
the c hemical c onjugation (or fusion) to cell penetrating
peptides (CPP) derived from proteins able to cross t he
plasma membrane. A large number of dierent c argo mol-

ecules such as oligonucleotides, peptides, peptide nucleic
acids, proteins or even nanoparticles have been internalized
in cells by this strategy. One of these translocating peptides
was derived from the HIV-1 Tat protein. The mechanisms by
which CPP enter cells remain unknown. Recently, convinc -
ing b iochemical and genetic ®ndings h as established that the
full-length Tat prote in was internaliz ed in ce lls via t he
ubiquitous heparan sulfate (H S) proteoglycans. We dem-
onstrate here that the short Tat CPP i s taken up by a route
that does not involve the HS proteoglycans.
Keywords: Tat; cell penetrating peptide (CPP); cellular
uptake; heparan sulfate.
Several cell-pe netrating peptides ( CPP) allowing the ef®cient
internalization o f various nonpermeant drugs in different
cell lines have been recently described. A covalent link had
to be created between the CPP and t he cargo molecule to
promote ef®cient membrane t ranslocation of the chimera
[1±7]. A 16-mer peptide derived from the Antennapedia
protein homeodomain [8] and a 13-mer peptide derived
from the HIV-1 Tat p rotein [9] have been extensively
studied. I n our initial experiments using the short Tat basic
domain, we demonstrated the uptake of chemically conju-
gated nonpermeant peptides [10]. Then, several peptides
showing a cellular activity w e re successfully vec torized either
with the Antennapedia p eptide [11] or the Tat peptide [12±
14]. Along the same lines, antisense oligonucleotides (ON)
were coupled chemically to the Antennapedia p eptide [1], or
to the short Tat peptide [2,15]. Ef®cient internalization and
biological activity of the O Ns were observed. Peptide nucleic
acids (PNAs) w ere also taken up by cells after coupling to

Transportan or to the Antennapedia peptide [3], or to the
Tat peptide (E. Vive
Á
s & B. Lebleu, unpublished observa-
tions). Regulation of the galanin receptor expression by a
sequence speci®c antisense activity was observed after
incubation of cells with the chimera [3]. The cellular
internalization of proteins such as b-galactosidase, horse-
radish peroxidase o r Fab antibody fragment was a lso
reported. In these cases, the carrier Tat peptide and the
transported protein were associated either by chemical
coupling [4,5,16] or by genetic construction leading to a
fusion protein expressing the 13-amino-acid CPP moiety a t
its N-terminus [6,7].
We have focused on the short HIV-1 Tat derived
peptide. Indeed it was initially shown that t he maximum
rate of inte rnalization was reached when three to four
molecules of a 35-amino-acid Tat peptide were ch emically
coupled to the transported protein [4]. In this case , the use
of shorter p eptides appeared to reduce the uptake process.
A structure±function relationship study of the peptide
encompassing this 35-amino-acid region then allowed
delineation of the t ranslocating activity domain t o a
13-mer amino-acid sequence [9]. This sequen ce contains
six arginine residues and two lysine residues within a linear
sequence of 13 amino acids, conferring a highly cationic
character on this peptide . It was later shown that arginine
residues were essential for translocation as deletion (or
replacement b y a lanine) of a s ingle arginine severely
reduced internalization [10,17].

The mechanism by which these cell penetrating peptides
(and their conjugates) e nter cells is not yet determined,
although endocytosis does not seem to be required [9,18].
First, it was shown for the Antennapedia peptide that
structural requirements were not involved in the uptake
process as the inverso
D
-isomer form of the peptide [19] or
insertion of proline residues w ithin the primary sequence
[18] did n ot impair cell uptake. Tat behaviour is very similar
to Antennapedia as the Tat peptide with all
D
-amino acids
(48GRKKRRQRRRPPQ60C) still enters cells [20] and the
retro-inverso form of the Tat peptide (57RRRQRRKKR49
with all
D
-amino acids) is even more ef®ciently translocated
than the corresponding native peptide [17]. Second, both
peptides are internalized at 4 °C [9,18], a temperature w hich
abolishes active transport mechanisms involving endocyto-
sis. Third, both peptides were found to be taken up in
Correspondence to E. V ives, Institut de Ge
Â
ne
Â
tique Mole
Â
culaire de
Montpellier, CNRS UMR 5124, BP5051, 1919 route de M ende, 34033

Montpellier cedex 1, France. Fax: + 33 467 040231,
Tel.: + 33 467 613661, E-mail:
Abbreviations: CPP, cell penetrating peptides; HS, heparan sulfate;
PNA, peptide nucleic acid; GST, gluthathione S-transferase; GFP,
green ¯uorescent prote in; FHV, ¯o ck hous e virus.
(Received 7 September 2001, accepted 14 November 2001)
Eur. J. Biochem. 269, 494±501 (2002) Ó FEBS 2002
various tissue types suggesting an ubiquitous process of
internalization w hich strongly suggests binding to conserved
cell membrane determinants. Recently, convincing bio-
chemical and genetic evidence suggested that the cell surface
heparan sulfate (HS) proteoglycan s, which are expressed in
most cell types, are responsible for the internalization of the
full-length Tat protein fused to glutathione S-transferase
(GST) a nd/or green ¯uorescen t protein (GFP) [21]. More-
over, m utations in the basic domain of T at abolished uptake
of these constructions [22] thus indicating that this domain i s
essential for binding to the receptor. The present work
aimed at de®ning whether membrane translocation of the
full-length Tat protein and cellular uptake o f its basic
domain make u se of the s ame mechanism. B oth g enetic and
biological evidence indicates that the cellular uptake of the
Tat basic peptide does not involve binding to HS proteo-
glycans and endocytosis.
EXPERIMENTAL PROCEDURES
Peptide synthesis and labeling
Peptide synthesis was performed by solid phase on a Pioneer
synthesizer (Applied Biosystems, Forster City, CA, USA)
following th e F moc chemistry protocol. The Tat peptide
sequence was Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-

Arg-Pro-Pro-Gln-Cys as previously described [10]. The
Cys residue was added to the C-terminal end of the
13-amino-acid peptide corresponding to the primary
sequence of the Tat protein to provide a sulfhydryl group
for further ligation to a ¯uorochrome or to a cargo
molecule. The peptide was puri®ed by semipreparative
HPLC and characterized by analytical HPLC, amino-acid
analysis and MALDI-TOF analysis. R esults were in full
agreement with the expected criteria (data not shown).
Labeling with the ¯uorochrome was performed on the
puri®ed Tat peptide through its cysteine side chain by
conjugation with a 10-fold molar excess of ¯uorescein
or rhodamine-maleimide derivatives (Molecular P robes
Europe BV, Leiden, the Netherlands) in 50 m
M
Tris/HCl
buffer pH 7.2 for 4 h in the dark. Labeled peptides were
puri®ed by semipreparative HPLC , freeze-dried, and resus-
pended in NaCl/P
i
at 1 mgámL
)1
. Peptide concentration
was a ssessed by quantitative a mino-acid analysis. Peptides
were st ored f rozen a t )20 °C until further use.
Cells and cell cultures
HeLa cells were cultured as exponentially growing subcon-
¯uent monolayers o n 90-mm plates in RPMI 1640 medium
(Gibco) supplemented with 10% (v/v) fetal bovine serum
and 2 m

M
glutamine. Wild-type CHO K1 cells and CHO
mutants de®cient in p roteoglycan biosynthesis [21] were
obtained from ATCC (Manassas, VA). T he A-745 and
D-677 mutant c ells were fully defective in proteoglycans.
The B-618 mutant produces about 15% of th e normal level
of the proteoglycans synthesized in wild-type. The E-606
mutant produces an undersulfated form of HS proteogly-
can. Finally, the C-605 mutant has also a defect in sulfate
uptake leading to low expression of wild-type HS proteo-
glycans. CHO cell lines were grown in D ulbecco's modi®ed
Eagle's medium (Gibco) supplemented with 10% (v/v) fetal
bovine serum.
Tat peptide internalization
Exponentially growing cells wer e dissociated with a
nonenzymatic cell dissociation medium (Sigma). C ells
(15 ´ 10
3
per well) were plated on eight-well LabTek
coverslips (Nunc Inc.) and cultured overnight. The culture
medium was discarded and the cells w ere washed with
NaCl/P
i
(pH 7 .3). Cells were preincubated in 100 lLof
Opti-MEM (Gibco) a t 37 °C for 30 min before incubation
with the peptide. Opti-MEM was discarded from the
coverslips and the cell m onolayers were incubated at
37 °C w ith Tat peptide dissolved in Opti-MEM at the
appropriate concentration. Subsequently, cells were rinsed
three times for 5 min with NaCl/P

i
(pH 7 .3) and ®xed in
3.7% (v/v) formaldehyde in NaCl/P
i
for 5 min at room
temperature. For experiments at 4 °C, the protocol was
the same except that all incubations were performed at
4 °C until the end of the ®xation procedure. For direct
detection of ¯uorescein-labeled or rhodamine-labeled
peptides, cells were washed three times after the ®xation,
then incubated with 50 n gámL
)1
of Hoechst 33258 in
NaCl/P
i
at room temperature, and washed again with
NaCl/P
i
before being processed in Vectashield
TM
mount-
ing solution (Vector Laboratories Inc., Burlingame, CA,
USA).
Internalization and detection of recombinant proteins
Recombinant GST±Tat protein and GST±Tat±GFP were
prepared as already described [21]. F or direct detection of
the GFP recombinant protein by ¯uorescence microscopy
the protocol was identical to Tat peptide internalization.
Incubation was performed at a protein concentration of
1 lgámL

)1
in the presence of 100 l
M
chloroquine in the cell
culture medium. For FACS analysis, the concentration of
the recombinant protein was increased to 5 lgámL
)1
.
The internalization of the GST±Tat co nstruct was
monitored by i mmunodetection as described previously
[21]. After incubation with the recombinant construct for
4 h , c ells were incubated with a monoclonal murine
antibody directed against the Tat 49±58 epitope (Hybrido-
lab, Institut Pasteur, Paris) at a ®nal concentration of
10 ng álL
)1
for 1 h at room t emperature. Cells were then
washed ®ve t imes for 5 min with warm NaCl/P
i
(25±28 °C)
before incubation with a rhodamine-conjugated anti-
(mouse IgG) Ig (Sigma) for 30 min. The distribution of
the ¯uorescence was analysed by microscopy on a Zeiss
Axiophot ¯uorescence microscope [9].
Flow cytometry
To analyze the intern alization of ¯uorochrome-labeled Tat
peptides or GFP-Tat by c ell c ytometry, 5 ´ 10
5
cells pe r well
were plated and c ultured overnight. The culture medium

was discarded, the cells were washed with NaCl/P
i
(pH 7.3)
andpreincubatedin1mLOpti-MEMat37°Cfor30min
before incubation with the ¯ uorescent c onstructs. C ells were
washed three times with NaCl/P
i
, dissociated with non-
enzymatic cell dissociation medium, centrifuged at 250 g
and resuspended in 500 lLNaCl/P
i
. Fluorescence analysis
was performed with a FACScan ¯uorescence-activated cell
sorter (Becton Dickinson). A total of 10 000 events per
sample were analyzed.
Ó FEBS 2002 Tat cell penetrating peptide uptake (Eur. J. Biochem. 269) 495
Cell treatment with heparinase III
Cell treatment with the heparinase III GAG lyase (Sigma)
was performed as previously described [21]. However for
easier hand ling of the cells, treatment was performed on
HeLa cells instead of CHO K1 cells. C ells were then
incubated with of 5 lgámL
)1
Tat±GFP f usion protein or
with 1 l
M
¯uorescein Tat peptide and analyzed by FACS.
RESULTS
Uptake and cellular localization of ¯uorescently
labeled Tat peptides

Cellular uptake of the full-length Tat p rotein fused to GFP
and/or GST involves a n interaction with cell surface HS
proteoglycans as recently demonstrated by biochemical a nd
genetic experiments [21]. To establish whe ther the short Tat
CPP follows the same internalization process, the ¯uores-
cein-labeled Tat peptide was incubated with the same cell
lines, namely wild-type (wt) CHO-K1 cells and A-745
mutant cells which are completely defective in HS sulfate
expression [21]. As a positive control, uptake of the Tat
peptide in HeLa cells was also monitored, as performed in
previous studies [9].
Uptake of the short ¯uorescein-labeled Tat peptide took
place in wt-CHO cells and in the A-745 cell line (Fig. 1; top
panels), thus indicating that internalization o f this short Tat
peptide does not require HS e xpression. The morphology of
CHO cells and their weak adherence on the glass slide
rendered subcellular l ocalization more dif®cult to assess
than in HeLa cells. However, a nucleolar concentration in
both CHO cell lines clearly took place (as indicated by
triangles in Fig. 1) in agreement with data previously
reported b y our laboratory [9]. Incubation of the Tat
peptide w as performed over a wide time range (from 15 min
to 24 h) and no major differences in intracellular distribu-
tion were observed (data no shown).
In order to exclude a possible in¯uence of the conjugated
¯uorochrome on translocation and intracellular distribu-
tion, the same experiments were performed with a Tat
peptide l abeled with rhodamine maleimide on i ts C-term inal
cysteine residue (Fig. 1; botto m panels) or on its N-terminal
residue (data not shown). No difference in the intracellular

distribution of the peptide was observed w hether wild-type
or mutants H S-de®cient CHO cells were used. Moreover
identical results showing internalization of ¯uorochrome
labeled peptide were obtained with the other HS mutated
cell lines described in Experimental procedures (data not
shown).
Flow cytometry analysis of the Tat peptide
internalization
Fluorescence microscop y clearly indicated internalization of
the ¯uorescent peptide in wild-type an d m utant HS de®cient
CHO c ell lines. W e t hen monitored t he internalization of the
Tat peptide by ¯ow cytometry analysis (Fig. 2), a technique
allowing the evaluation of the homogeneity of the cellular
population i n terms of uptake e f®ciency. As previously
Fig. 1. Fluorescence microscopy analysis of Tat p eptide uptake in HS expression de®cient cell lines. HS expressing (HeLa, wt-CHO ) or de®cient
(CHO A-745) cell lines were incubated with ¯u orescein-labeled T at (top pan els) or with rh odamine-labeled T at (bo ttom panels) for 1 5 min at 37 °C.
Uptake and intracellular distribution were monitored by ¯u orescenc e microscopy with the ap propriate ®lters. Small triangles indicate the n ucle ol ar
concentration of peptides in the dierent cell lines.
496 M. Silhol et al. (Eur. J. Biochem. 269) Ó FEBS 2002
observed b y ¯uorescence microscopy, the internalization of
the Tat peptide took place to the same extent in HeLa cells,
in wt-CHO ce lls and in t he A-745 (defective i n HS
proteoglycan) mutant cell line. Moreover internalization
appeared to be homogeneous in the w hole cell population as
a s ingle massif was observe d for all c ell lines (Fig. 2A). In
order t o minimize cell handling prior to FACS analysis, no
®xation step was included. Avoiding cell ®xation and
working on living cells eliminates potential artefacts linked
with cell processing. FACS analysis showed that cellular
uptake and distribution of the peptide was identical in ®xed

cells or in living cells (data not shown) in agreement with
previous data on other cell lines [9] and with ¯uorescence
microscopy data reported above.
To avoid any possible artefactual data in handling the
different cell lines and/or experimental conditions, we
reproduced the published results on the internalization of
the T at protein fusion construct [21]. In k eeping with
previous work [21], the full-length Tat protein tested as a
fusion recombinant protein with GST and GFP w as
normally internalized in wild-type cell line while the uptake
was markedly inhibited o n A-745 HS proteoglycans
de®cient cells (Fig. 2B).
The uptake of Tat CPP w as further examined by FACS
analysis in dose±response e xperiments at peptide concen-
trations ranging from 1 00 n
M
to 10 l
M
for 15 min incuba-
tion time (Fig. 3). This was performed on HeLa cells in
which uptake of the fused Tat protein has been shown to
involve HS proteoglycans [21]. A saturation of the ¯uores-
cent signal was observed for extracellular doses above 1 l
M
.
Whether this could re¯ect a saturation of the potential
cellular binding sites for the peptide was not fully investi-
gated. Along the s ame lines, competition exper iments
between a ®xed dose of ¯uorescein-Tat peptide (100 n
M

)
and increasing doses of unlabeled Tat peptide (u p to
100 l
M
) only led to a slight reduction of the intracellular
signal (data not shown). Whether there is saturation of
intracellular binding sites or competition at the level of
membrane structu res implicated in the T at peptide uptake i s
under evaluation.
Comparative FACS analysis of the internalization
of the full-length Tat protein construct and the Tat CPP
Differences in the mechanisms of internalization between
the Tat peptide a nd the Tat fused p rotein was a lso
established by adding the Tat±GFP construct w ith the
rhodamine-labeled Tat CPP in competition. The internal-
ization of the Tat protein fused to GFP was detected by
recording t he intensity o f the GFP signal itself in the 440 nm
wavelength range (Fig. 4). Rhodamine-labeled Tat peptide
internalization was monitored in the 560 nm wavelength
range (data n ot sh own). The Tat±GFP was incubated w ith
wt-CHO in the absence (bold line) or in the p resen ce ( dotted
Fig. 3. Dose±response study of Tat peptide uptake in HeLa cells by
FACS analysis. HeLa ce lls were incubate d with i ncreasing amounts of
the rhodamine-labeled Tat peptide, as indicated in the ®gure.
Fig. 2. FACS analysis of Tat peptide and
Tat±GFP fusion protein uptake in HS
expressing or de®cient cell lines. Plain lines
in all panels correspond to untreated cells.
(A) HS expressing (HeLa, wt-CHO) or de®-
cient (CHO A-745) cell lines were incuba ted

with 10 l
M
¯uorescein labeled Tat peptide
(dotted lines). (B) A s a control, HS-expressing
(wt-CHO, left frame) or de®cient (CHO
A-745, right frame) cell lines were incubated
for 4 h at 37 °C with T at±GF P fusion protein
(bold lines).
Ó FEBS 2002 Tat cell penetrating peptide uptake (Eur. J. Biochem. 269) 497
line) of a 12.5-fold molar excess o f t he rhodamine-Tat
peptide competitor (80 n
M
and 1 l
M
, respectively). As
shown in Fig. 4 (panel A), the internalization of the Tat±
GFP fusion construct was not signi®cantly reduced in the
presence of the excess of t he Tat peptide, in keep ing with
separate internalization pathways. Internalization of the
Tat±GFP fusion construct in these conditions was poorly
ef®cient in the A-745 clone (Fig. 4, Panel B) as previously
described. A weak displacement of the p eak detected in the
¯uorescein channel c ould be due to nonreceptor mediated
endocytosis during the 24 h incubation time.
Differences in the uptak e mechanism between the two
Tat entities w ere also c on®rmed by t he temperature
dependence of the internalization process. As shown in
Fig. 5, ¯uorescein-labeled Tat peptide internalization was
not abolished by low temperature (dotted lines in Fig. 5, left
and right panels) in keeping with our previous data [9].

However a rightward shift of the s ignal was observed
signifying a reduction of the uptake of the Tat peptide at
low temperature. Likewise, a threefold r eduction of the
uptake at 4 °C has been reported for the Antennapedia
peptide compared to i ts cellular uptake at 3 7 °C [23]. At
variance with the Tat±GFP fusion construct (bold line in
Fig. 5 left and right), the ¯uorescent signal was completely
inhibited as expected for HS proteoglycans-mediated end-
ocytosis.
In order t o c on®rm t he involvment of HS receptors i n t he
uptake of the Tat protein, HeLa cells were treated with
heparinase III, an enzyme mostly active o n HS proteogly-
cans [21]. The uptake of full length Tat protein was
abolished b y such t reatment on CHO K1 cells [21].
Likewise, heparinase treatment of HeLa cells completely
inhibits the uptake of the Tat±GFP f usion protein (Fig. 6A,
dotted line). On the contrary, the internalization of the Tat
peptide was not affected by the heparinase treatment
(Fig. 6 B, dotted line) as similar internalized ¯uoresc ence
was quanti®ed in heparinase-treated cells compared to
untreated cells (Fig. 6B, bold line).
DISCUSSION
Intracellular v ectorization after chemical coupling or genetic
fusion to the CPP derived from the HIV-1 Tat appears as a
potent tool for the cellular delivery of various biomolecules.
These include oligonucleotides [2], peptides [10±12,14],
proteins [4,6,7], nanoparticles [24] or liposomes [25]. The
internalization process is not cell speci®c as a large number
of cell lines tested so far entrapped the translocating peptide.
Fig. 4. Competition between the T at-peptide and the Tat±GFP fusion prot ein in HS expressing or de®cient cells. (A) H S expressing cells were

coincubated for 24 h w ith the Tat-rho damin e peptide a nd the T at±GFP fusion prote in. The u ptake of t he Tat±GFP f usion protein was monitored
in the absence (bold line) or in the presence (dotted line) o f competitor Tat-rhodam ine peptide. Uptake was monitored by FACS an alysis in the
green channel to account for T at±GFP fusion protein uptake. (B) HS de®cient A-745 c ells were incubated in identical conditions with bo th Tat
entities. FACS analysis was monitored in the green channel. Signal record in the red channel showed strong cellular labeling (not shown). Plain lines
in both ®gures corresponds to untre ated cells.
Fig. 5. In¯uen ce of tem perat ure on the up take
of Tat CPP and of Tat±GFP fusion protein.
HeLa cells were incubated during 4 h with
Tat±GFP (solid lines) or with ¯uorescein-
labeled Tat peptide (dotted lines) at 37 °C
(leftpanel)orat4°C (right panel). Uptake
was monitored by FACS analysis.
498 M. Silhol et al. (Eur. J. Biochem. 269) Ó FEBS 2002
These include cell types which were very poorly transfected
by traditional methods as monocyte/macrophages progen-
itors [26]. Moreover Tat peptide conjugated molecules also
pass through the blood brain barrier [6].
Despite the large number of potential applications of
these CPP, the mechanism by which translocation proceeds
remains essentially unknown. Interestingly, HS proteogly-
cans we re recently shown to be responsible for the uptake o f
the Tat protein in a large number o f cell lines [21]. The
present studies were designed to test whether t he short
HIV-1 Tat peptide could e nter ce lls via this receptor t ype.
We ®rst made use of CHO mutant cell lines de®cient in th e
expression of HS proteoglycans [21]. We clearly established
that the ¯uorochrome labeled Tat CPP was taken up in
these mutant cell lines as ef®ciently than in wt-CHO or in
HeLa cells. T he internalization of the peptide i n these c ell
lines was monitored in parallel by ¯uorescence microscopy

and by FACS s can analysis. The ® rst technique con®rmed
the uptake of the peptide an d its nucleo lar concentration in
CHO cells as previously observed in HeLa cells [9]. The
second technique showed that all the cells from a nonsyn-
chronized population entrapped the peptide a lthough the
¯uorescence intensity could be slightly variable among that
population. In addition to these genetic ®ndings, we treated
cells w ith h eparinase III prior to their incubation with the
different Tat derived molecules in order to digest HS
receptors. As previously described [21], such treatment
abolished the internalization of the GFP-fused Tat protein
but did not alter the uptake of the Tat CPP. These
biochemical evidences con®rmed a pathway for the e ntry of
the Tat peptide unrelated to the HS proteoglycan receptors.
Internalization of the Tat CPP did not use a classic
endocytosis pathway either, a s low temperature i ncubation
of the cells did not impair dramatically the Tat peptide
uptake while it abolished the uptake of the GFP-fused Tat
protein as expected. Translocation at low temperature was
initially described for the Tat peptide [9] and for the
Antennapedia peptide [8]. However, a reduction of the Tat
peptide uptake could be observed in our experiments when
comparing FACS s ignal intensity at 4 and at 37 °C (Fig. 5).
An identical reduction of the uptake at 4 °C was recently
reported for the Antennapedia peptide as well [23]. Even
reduced, unambiguous internalization of both peptides at
low temperature indicates the existence of an endocytosis
independant process for cellular entry. Low temperature
translocation of co njugated molecules w as recently o bserved
to be also effective a s published f or liposomes attach ed with

the short T at peptide [25]. Moreover in our experiments, the
rhodamine labeled Tat peptide was coincubated with the
GFP-Tat fusion protein to assess the effective inhibition of
the receptor mediated endocytosis. Despite a 12.5 molar
excess of the Tat peptide, no detectable reduction of the
uptake of the Tat±GFP fusion protein was observed when
cells were incubated at 37 °C, thus providing additional
evidences for separate entry routes for the Tat CPP and the
Tat protein.
What might be the reasons underlying the observed
differences in cellular uptake between the Tat CPP and the
GST±Tat±GFP protein, that both contain the same amino-
acid sequence? It might be envisaged that the Tat basic
domain is found in different molecular environments in the
two molecular species. In the case of the short Tat CPP, the
cluster of b asic amino acids is likely to be fully ac cessible to
cellular components inducing the translocation event, with
particular reference to the arginine residues which appear
to be the main determinants for the translocating activity
[10,17]. Within the large recombinant protein, t he exposure
and/or the environment of t his basic cluster of amino acids
might be different, even if the high hydrophilic nature of
this domain likely leads to its exposure at the surface of the
GST±Tat fusion protein as it does in the Tat protein itself
[27]. Easy accessibility of this domain can be also inferred
from the notion that both a GST±Tat and a GST±Tat±
GFP fusion proteins are able to transactivate the HIV-1
LTR sequence, an event which requires binding of the Tat
basic domain to the TAR sequence on nascent RNAs
[21,28,29]. Accordingly, no t ransactivation was obtained

when the argin ine residues from the Tat basic domain w ere
mutated to alanine in a HeLa derived cell line [21]. These
considerations indirectly reinforce the argument that the
basic domain should be exposed at the surface of the
Tat-containing recombinant proteins and call other reasons
to explain the differences in the mechanism of internaliza-
tion between the CPP peptide and the Tat-containing
proteins. A long this line, it has been reported that chemical
coupling of T at peptides with different length to hetero-
logous proteins resulted in variable ef®ciency of internali-
zation [4]. In particular, it was reported that the maximum
rate of internalization was reached when three or four
molecules of a 35-amino-acid Tat peptide (sequence 37±72)
were chemically coupled to a large protein cargo. Despite
the presence of the basic region (sequence 49±57), the use
of shorter chemically-bound peptides (sequence 37±58 or
47±58) was described to be less effective than the Tat
Fig. 6. In¯uence of heparinase III treatment
on the uptake of Tat CPP and of Tat±GFP
fusion protein. HeLa cells were incubated w ith
5 lgámL
)1
Tat±GFP fusion protein or with
1 l
M
¯uorescein Tat peptide. (A) Incubation
of the cell with T at±GFP fusion protein
without (plain line) or with heparinase treat-
ment (dotted line). (B) Incubation of the cell
with ¯uorescein Tat CPP without (plain line)

or with heparinase treatment (dotted line).
Uptake was monitored by FACS analysis.
Ó FEBS 2002 Tat cell penetrating peptide uptake (Eur. J. Biochem. 269) 499
peptide 37±72 in the internalization process [4]. Thus, steric
hindrance of the heterologous protein itself could reduce
the exposure of these shorter peptides to cellular structures,
and therefore, reduce the ef®ciency of translocation. For
recombinant fusion proteins, it has been clearly demon-
strated that an 11-amino-acid peptide containing only the
basic a mino-acid cluster is highly ef®cient in mediating
internalization of heterologou s proteins w hen fused at the
N-terminal domain of these proteins [6]. Cellular internal-
ization of this peptide fused to b-galactosidase was even
observed in vivo in various tissues including the brain after
intraperitoneal injection into the mouse [6]. While com-
parative studies are still lacking, it can be speculated that
fusion of the Tat peptide to t he N-terminal region of
proteins favors its steric accessibility to cellular structures
involved in the translocation process, thus accounting for
the more ef®cient cellular internalization of these fusion
proteins as compared to th eir chemically linked counter-
parts. This would explain why a fusion con struct contain -
ing only one Tat peptide sequence at its N-terminal end i s
taken up more e f®ciently than chemically linked b-galacto-
sidase despite the higher number of peptides. As far as
Tat peptides are concerned, the 13-amino-acid peptide
encompassing the basic domain of Tat (Tat 48±60) was
found to be more effective than longer peptides such as Tat
43±60 or Tat 37±60 [9]. The primary sequence of the Tat
peptide itself does not seem to be a key feature in cell

uptake as several analogues were tested without noticeable
variation of the cellular uptake intensity provided the total
number of basic amino acids was l eft unchanged (E. Vive
Á
s
&B.Lebleuet al. unpublished results). Likewise the retro-
inverso form of the p eptide did not impair the Tat
translocating properties [17,20]. A receptor-mediated mech -
anism of cellular i nternalization of the peptide thus appears
unlikely. The number of arginine residues within the Tat
peptide appeared to be the main determinant for main-
taining a high translocating activity as pre viously shown by
alanine-arginine substitution scan [10,17]. Several other
arginine-rich p eptides, such as ¯ock house virus (FHV) or
Rev derived peptides, showed similar cell up take p roperties
[20]. It was shown recently that short polyarginine p eptides
were even more potently internalized into cells [17,20].
Moreover the length of the polyarginine tract seems
critical, as a maximal rate of internalization was observed
for a peptide nine arginine residues i n length. The
D
-form
and the retro-inverso form of the polyarginine peptide
were found to internalize more e f®ciently. However the
higher stability in serum containing cell culture m edium of
the
D
-form or t he peptides was proposed as the reason of
this apparent increased uptake, as the rate of uptake was
thesameinserum-freemedium[17].Asalreadystated

above, this Tat CPP peptide is able to vectorize various
cargo molecules inside cells [2,4±7,10,12±16]. Strikingly,
ef®cient internalization in vitro and in vivo of ferromagnetic
particles (45 nm diameter) when three to four short Tat
peptide molecules were conjugated to it [24] suggests a
noncommon mechanism of entry. Whether binding to
other cell surface determinants (as for instance to polar
lipid heads) is involved is currently be ing investigated.
Whatever the mechanism however, the possibility to deliver
heterologous molecules into different tissues and even
through the blood brain b arrier has high potential in
biotechnology.
ACKNOWLEDGEMENTS
We thank Dr Pierre Travo for his help in ¯uorescence imagi ng and
computerized analysis of pictures. We are grateful to D r Jean-Jacques
Vasseur f or performing MALDI-TOF an alysis o f peptides. We also
thank I. Robbins for proofreading of the manuscript. This work was
supported by grants from the Association pour la Recherche sur le
Cancer to B. L. and E. V. and from MURST a nd Istituto Superiore di
Sanita¢,Rome,ItalytoM.G.
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