Different mechanisms for cellular internalization of the HIV-1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat Michelle Silhol’, Mudit Tyagi, Mauro Giacca’,
Trang 1Different mechanisms for cellular internalization of the HIV-1
Tat-derived cell penetrating peptide and recombinant proteins
fused to Tat
Michelle Silhol’, Mudit Tyagi, Mauro Giacca’, Bernard Lebleu’ and Eric Vives'
‘Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5124, BP5051, Montpellier, France;
? 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 chemical conjugation (or fusion) to cell penetrating
peptides (CPP) derived from proteins able to cross the
plasma membrane A large number of different cargo 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 biochemical and genetic findings has established that the full-length Tat protein was internalized in cells via the ubiquitous heparan sulfate (HS) proteoglycans We dem- onstrate here that the short Tat CPP is taken up by a route that does not involve the HS proteoglycans
Keywords Tat; cell penetrating peptide (CPP); cellular uptake; heparan sulfate
Several cell-penetrating peptides (CPP) allowing the efficient
internalization of various nonpermeant drugs in different
cell lines have been recently described A covalent link had
to be created between the CPP and the cargo molecule to
promote efficient membrane translocation 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 protein [9] have been extensively
studied In 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 were successfully vectorized either
with the Antennapedia peptide [11] or the Tat peptide [12—
14] Along the same lines, antisense oligonucleotides (ON)
were coupled chemically to the Antennapedia peptide [1], or
to the short Tat peptide [2,15] Efficient internalization and
biological activity of the ONs were observed Peptide nucleic
acids (PNAs) were also taken up by cells after coupling to
Transportan or to the Antennapedia peptide [3], or to the
Tat peptide (E Vivés & B Lebleu, unpublished observa-
tions) Regulation of the galanin receptor expression by a
sequence specific antisense activity was observed after
incubation of cells with the chimera [3] The cellular
internalization of proteins such as B-galactosidase, horse-
radish peroxidase or Fab antibody fragment was also
reported In these cases, the carrier Tat peptide and the
Correspondence to E Vives, Institut de Génétique Moléculaire de
Montpellier, CNRS UMR 5124, BP5051, 1919 route de Mende, 34033
Montpellier cedex 1, France Fax: + 33 467 040231,
Tel.: + 33 467 613661, E-mail: vives@igm.cnrs-mop.fr
Abbreviations: CPP, cell penetrating peptides; HS, heparan sulfate;
PNA, peptide nucleic acid; GST, gluthathione S-transferase; GFP,
green fluorescent protein; FHV, flock house virus
(Received 7 September 2001, accepted 14 November 2001)
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 at its N-terminus [6,7]
We have focused on the short HIV-1 Tat derived peptide Indeed it was initially shown that the maximum rate of internalization was reached when three to four molecules of a 35-amino-acid Tat peptide were chemically coupled to the transported protein [4] In this case, the use
of shorter peptides 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 translocating activity domain to a
13-mer amino-acid sequence [9] This sequence 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 by alanine) of a single arginine severely reduced internalization [10,17]
The mechanism by which these cell penetrating peptides (and their conjugates) enter 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 within the primary sequence [18] did not impair cell uptake Tat behaviour is very similar
to Antennapedia as the Tat peptide with all p-amino acids (48GRKKRRQRRRPPQ60C) still enters cells [20] and the retro-inverso form of the Tat peptide (S7RRRQRRKKR49 with all p-amino acids) is even more efficiently translocated than the corresponding native peptide [17] Second, both peptides are internalized at 4 °C [9,18], a temperature which abolishes active transport mechanisms involving endocyto- sis Third, both peptides were found to be taken up in
Trang 2© FEBS 2002
various tissue types suggesting an ubiquitous process of
internalization which strongly suggests binding to conserved
cell membrane determinants Recently, convincing bio-
chemical and genetic evidence suggested that the cell surface
heparan sulfate (HS) proteoglycans, which are expressed in
most cell types, are responsible for the internalization of the
full-length Tat protein fused to glutathione S-transferase
(GST) and/or green fluorescent protein (GFP) [21] More-
over, mutations in the basic domain of Tat abolished uptake
of these constructions [22] thus indicating that this domain is
essential for binding to the receptor The present work
aimed at defining whether membrane translocation of the
full-length Tat protein and cellular uptake of its basic
domain make use of the same mechanism Both genetic 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 the Fmoc 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 fluorochrome or to a cargo
molecule The peptide was purified by semipreparative
HPLC and characterized by analytical HPLC, amino-acid
analysis and MALDI-TOF analysis Results were in full
agreement with the expected criteria (data not shown)
Labeling with the fluorochrome was performed on the
purified Tat peptide through its cysteine side chain by
conjugation with a 10-fold molar excess of fluorescein
or rhodamine-maleimide derivatives (Molecular Probes
Europe BV, Leiden, the Netherlands) in 50 mm Tris/HCl
buffer pH 7.2 for 4 h in the dark Labeled peptides were
purified by semipreparative HPLC, freeze-dried, and resus-
pended in NaCl/P; at 1 mgmL7! Peptide concentration
was assessed by quantitative amino-acid analysis Peptides
were stored frozen at —20 °C until further use
Cells and cell cultures
HeLa cells were cultured as exponentially growing subcon-
fluent monolayers on 90-mm plates in RPMI 1640 medium
(Gibco) supplemented with 10% (v/v) fetal bovine serum
and 2 mm glutamine Wild-type CHO K1 cells and CHO
mutants deficient in proteoglycan biosynthesis [21] were
obtained from ATCC (Manassas, VA) The A-745 and
D-677 mutant cells were fully defective in proteoglycans
The B-618 mutant produces about 15% of the 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 Dulbecco’s modified
Eagle’s medium (Gibco) supplemented with 10% (v/v) fetal
bovine serum
Tat cell penetrating peptide uptake (Eur J Biochem 269) 495
Tat peptide internalization Exponentially growing cells were dissociated with a nonenzymatic cell dissociation medium (Sigma) Cells (15x 10° per well) were plated on eight-well LabTek coverslips (Nunc Inc.) and cultured overnight The culture medium was discarded and the cells were washed with NaCl/P; (pH 7.3) Cells were preincubated in 100 uL of Opti-MEM (Gibco) at 37 °C for 30 min before incubation with the peptide Opti- MEM was discarded from the coverslips and the cell monolayers were incubated at
37 °C with Tat peptide dissolved in Opti-MEM at the
appropriate concentration Subsequently, cells were rinsed
three times for 5 min with NaCl/P; (pH 7.3) and fixed in 3.7% (v/v) formaldehyde in NaCl/P; 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 fixation procedure For direct detection of fluorescein-labeled or rhodamine-labeled peptides, cells were washed three times after the fixation, then incubated with 50 ngmL7! of Hoechst 33258 in NaCl/P; at room temperature, and washed again with NaCl/P; before being processed in Vectashield™ 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] For direct detection of the GFP recombinant protein by fluorescence microscopy the protocol was identical to Tat peptide internalization Incubation was performed at a protein concentration of
1 ngmL” in the presence of 100 1m chloroquine in the cell culture medium For FACS analysis, the concentration of the recombinant protein was increased to 5 ugmL™ The internalization of the GST-—Tat construct was monitored by immunodetection as described previously [21] After incubation with the recombinant construct for
4h, cells were incubated with a monoclonal murine
antibody directed against the Tat 49-58 epitope (Hybrido-
lab, Institut Pasteur, Paris) at a final concentration of
10 nguL7! for 1 h at room temperature Cells were then washed five times for 5 min with warm NaCl/P; (25-28 °C) before incubation with a rhodamine-conjugated anti- (mouse IgG) Ig (Sigma) for 30 min The distribution of the fluorescence was analysed by microscopy on a Zeiss
Axiophot fluorescence microscope [9]
Flow cytometry
To analyze the internalization of fluorochrome-labeled Tat peptides or GFP-Tat by cell cytometry, 5 x 10° cells per well were plated and cultured overnight The culture medium was discarded, the cells were washed with NaCl/P; (pH 7.3) and preincubated in 1 mL Opti-MEM at 37 °C for 30 min before incubation with the fluorescent constructs Cells were
washed three times with NaCl/P;, dissociated with non-
enzymatic cell dissociation medium, centrifuged at 250 g and resuspended in 500 uL NaCl/P; Fluorescence analysis was performed with a FACScan fluorescence-activated cell sorter (Becton Dickinson) A total of 10 000 events per sample were analyzed
Trang 3Cell treatment with heparinase III
Cell treatment with the heparinase II] GAG lyase (Sigma)
was performed as previously described [21] However for
easier handling of the cells, treatment was performed on
HeLa cells instead of CHO KI cells Cells were then
incubated with of 5 p;gemL~! Tat-GFP fusion protein or
with | um fluorescein Tat peptide and analyzed by FACS
RESULTS
Uptake and cellular localization of fluorescently
labeled Tat peptides
Cellular uptake of the full-length Tat protein fused to GFP
and/or GST involves an interaction with cell surface HS
proteoglycans as recently demonstrated by biochemical and
genetic experiments [21] To establish whether the short Tat
CPP follows the same internalization process, the fluores-
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 fluorescein-labeled Tat peptide took
place in wt-CHO cells and in the A-745 cell line (Fig 1; top
panels), thus indicating that internalization of this short Tat
peptide does not require HS expression The morphology of
CHO cells and their weak adherence on the glass slide
rendered subcellular localization more difficult 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 by our laboratory [9] Incubation of the Tat peptide was 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 influence of the conjugated fluorochrome on translocation and intracellular distribu- tion, the same experiments were performed with a Tat peptide labeled with rhodamine maleimide on its C-terminal cysteine residue (Fig 1; bottom panels) or on its N-terminal residue (data not shown) No difference in the intracellular distribution of the peptide was observed whether wild-type
or mutants HS-deficient CHO cells were used Moreover identical results showing internalization of fluorochrome 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 microscopy clearly indicated internalization of the fluorescent peptide in wild-type and mutant HS deficient CHO cell lines We then monitored the internalization of the Tat peptide by flow cytometry analysis (Fig 2), a technique allowing the evaluation of the homogeneity of the cellular population in terms of uptake efficiency As previously
A-745
Fig 1 Fluorescence microscopy analysis of Tat peptide uptake in HS expression deficient cell lines HS expressing (HeLa, wt-CHO) or deficient (CHO A-745) cell lines were incubated with fluorescein-labeled Tat (top panels) or with rhodamine-labeled Tat (bottom panels) for 15 min at 37 °C Uptake and intracellular distribution were monitored by fluorescence microscopy with the appropriate filters Small triangles indicate the nucleolar concentration of peptides in the different cell lines.
Trang 4© FEBS 2002 Tat cell penetrating peptide uptake (Eur J Biochem 269) 497
100 100
Fig 2 FACS analysis of Tat peptide and
expressing or deficient cell lines Plain lines
80 LL 80
20 L 20 Lai
A-745
in all panels correspond to untreated cells
(A) HS expressing (HeLa, wt-CHO) or defi-
cient (CHO A-745) cell lines were incubated
with 10 um fluorescein labeled Tat peptide
(dotted lines) (B) As a control, HS-expressing
(wt-CHO, left frame) or deficient (CHO
A-745, right frame) cell lines were incubated
z0 _
10
observed by fluorescence microscopy, the internalization of
the Tat peptide took place to the same extent in HeLa cells,
in wt-CHO cells and in the A-745 (defective in HS
proteoglycan) mutant cell line Moreover internalization
appeared to be homogeneous in the whole cell population as
a single massif was observed for all cell lines (Fig 2A) In
order to minimize cell handling prior to FACS analysis, no
fixation step was included Avoiding cell fixation 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 1n fixed
cells or in living cells (data not shown) 1n agreement with
previous data on other cell lines [9] and with fluorescence
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 Tat protein fusion construct [21] In keeping with
previous work [21], the full-length Tat protein tested as a
fusion recombinant protein with GST and GFP was
normally internalized in wild-type cell line while the uptake
was markedly inhibited on A-745 HS _ proteoglycans
deficient cells (Fig 2B)
The uptake of Tat CPP was further examined by FACS
analysis in dose-response experiments at peptide concen-
trations ranging from 100 nm to 10 uM 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 fluores-
cent signal was observed for extracellular doses above | LM
Whether this could reflect a saturation of the potential
cellular binding sites for the peptide was not fully investi-
gated Along the same lines, competition experiments
between a fixed dose of fluorescein-Tat peptide (100 nm)
and increasing doses of unlabeled Tat peptide (up to
100 um) 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 structures implicated 1n the Tat peptide uptake 1s
under evaluation
Fig 3 Dose-response study of Tat peptide uptake in HeLa cells by FACS analysis HeLa cells were incubated with increasing amounts of the rhodamine-labeled Tat peptide, as indicated in the figure
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 and the Tat fused protein was also established by adding the Tat-GFP construct with the rhodamine-labeled Tat CPP in competition The internal- ization of the Tat protein fused to GFP was detected by recording the intensity of 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 not shown) The Tat-GFP was incubated with wt-CHO 1n the absence (bold line) or in the presence (dotted
Trang 5
20 Ly
FL1-H
FL1-H
iw 108
10Ì
Fig 4 Competition between the Tat-peptide and the Tat—GFP fusion protein in HS expressing or deficient cells (A) HS expressing cells were coincubated for 24 h with the Tat-rhodamine peptide and the Tat-GFP fusion protein The uptake of the Tat-GFP fusion protein was monitored
in the absence (bold line) or in the presence (dotted line) of competitor Tat-rhodamine peptide Uptake was monitored by FACS analysis in the green channel to account for Tat-GFP fusion protein uptake (B) HS deficient A-745 cells were incubated in identical conditions with both 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 figures corresponds to untreated cells
line) of a 12.5-fold molar excess of the rhodamine-Tat
peptide competitor (80 nm and 1 uM, respectively) As
shown in Fig 4 (panel A), the internalization of the Tat—
GFP fusion construct was not significantly reduced in the
presence of the excess of the Tat peptide, in keeping with
separate internalization pathways Internalization of the
Tat-GFP fusion construct in these conditions was poorly
efficient in the A-745 clone (Fig 4, Panel B) as previously
described A weak displacement of the peak detected in the
fluorescein channel could be due to nonreceptor mediated
endocytosis during the 24 h incubation time
Differences in the uptake mechanism between the two
Tat entities were also confirmed by the temperature
dependence of the internalization process As shown 1n
Fig 5, fluorescein-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 signal was observed
signifying a reduction of the uptake of the Tat peptide at
low temperature Likewise, a threefold reduction of the
uptake at 4 °C has been reported for the Antennapedia
peptide compared to its cellular uptake at 37 °C [23] At
variance with the Tat-GFP fusion construct (bold line In
Fig 5 left and night), the fluorescent signal was completely
37°C 4°C
inhibited as expected for HS proteoglycans-mediated end- ocytosis
In order to confirm the involvment of HS receptors in the uptake of the Tat protein, HeLa cells were treated with heparinase III, an enzyme mostly active on HS proteogly- cans [21] The uptake of full length Tat protein was abolished by such treatment on CHO KI cells [21] Likewise, heparinase treatment of HeLa cells completely inhibits the uptake of the Tat—GFP fusion protein (Fig 6A, dotted line) On the contrary, the internalization of the Tat peptide was not affected by the heparinase treatment (Fig 6B, dotted line) as similar internalized fluorescence was quantified in heparinase-treated cells compared to untreated cells (Fig 6B, bold line)
DISCUSSION Intracellular vectorization 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 1s not cell specific as a large number
of cell lines tested so far entrapped the translocating peptide
of Tat CPP and of Tat—GFP fusion protein HeLa cells were incubated during 4 h with
=m a i | Tat—GFP (solid lines) or with fluorescein- labeled Tat peptide (dotted lines) at 37 °C
was monitored by FACS analysis.
Trang 6© FEBS 2002 Tat cell penetrating peptide uptake (Eur J Biochem 269) 499
Fig 6 Influence of heparinase III treatment
on the uptake of Tat CPP and of Tat-GFP
fusion protein HeLa cells were incubated with
5 ug-mL~' Tat-GFP fusion protein or with
1 um fluorescein Tat peptide (A) Incubation
of the cell with Tat-GFP fusion protein
without (plain line) or with heparinase treat-
ment (dotted line) (B) Incubation of the cell
with fluorescein Tat CPP without (plain line)
or with heparinase treatment (dotted line)
Uptake was monitored by FACS analysis
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 were recently shown to be responsible for the uptake of
the Tat protein in a large number of cell lines [21] The
present studies were designed to test whether the short
HIV-1 Tat peptide could enter cells via this receptor type
We first made use of CHO mutant cell lines deficient in the
expression of HS proteoglycans [21] We clearly established
that the fluorochrome labeled Tat CPP was taken up in
these mutant cell lines as efficiently than in wt-CHO or in
HeLa cells The internalization of the peptide in these cell
lines was monitored in parallel by fluorescence microscopy
and by FACS scan analysis The first technique confirmed
the uptake of the peptide and its nucleolar 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 although the
fluorescence intensity could be slightly variable among that
population In addition to these genetic findings, we treated
cells with heparinase 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 confirmed a pathway for the entry of
the Tat peptide unrelated to the HS proteoglycan receptors
Internalization of the Tat CPP did not use a classic
endocytosis pathway either, as low temperature incubation
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 signal 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 conjugated molecules was recently observed
to be also effective as published for liposomes attached with
the short Tat peptide [25] Moreover 1n 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 1s found in different molecular environments in the two molecular species In the case of the short Tat CPP, the cluster of basic amino acids 1s likely to be fully accessible 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, the exposure and/or the environment of this 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-—Yat 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—Fat 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 transactivation was obtained
when the arginine residues from the Tat basic domain were 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 Along this line, 1t has been reported that chemical coupling of Tat peptides with different length to hetero- logous proteins resulted in variable efficiency 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 4758) was described to be less effective than the Tat
Trang 7peptide 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 efficiency of translocation For
recombinant fusion proteins, it has been clearly demon-
strated that an 1l-amino-acid peptide containing only the
basic amino-acid cluster is highly efficient in mediating
internalization of heterologous proteins when 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 the N-terminal region of
proteins favors its steric accessibility to cellular structures
involved in the translocation process, thus accounting for
the more efficient cellular internalization of these fusion
proteins as compared to their chemically linked counter-
parts This would explain why a fusion construct contain-
ing only one Tat peptide sequence at its N-terminal end is
taken up more efficiently 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 left unchanged (E Vivés
& B Lebleu et al unpublished results) Likewise the retro-
inverso form of the peptide did not impair the Tat
translocating properties [17,20] A receptor-mediated mech-
anism of cellular internalization 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 previously shown by
alanine-arginine substitution scan [10,17] Several other
arginine-rich peptides, such as flock house virus (FHV) or
Rev derived peptides, showed similar cell uptake properties
[20] It was shown recently that short polyarginine peptides
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 in length The p-form
and the retro-inverso form of the polyarginine peptide
were found to internalize more efficiently However the
higher stability in serum containing cell culture medium of
the p-form or the peptides was proposed as the reason of
this apparent increased uptake, as the rate of uptake was
the same in serum-free medium [17] As already stated
above, this Tat CPP peptide is able to vectorize various
cargo molecules inside cells [2,4~-7,10,12—16] Strikingly,
efficient 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 being investigated
Whatever the mechanism however, the possibility to deliver
heterologous molecules into different tissues and even
through the blood brain barrier has high potential in
biotechnology
ACKNOWLEDGEMENTS
We thank Dr Pierre Travo for his help in fluorescence imaging and computerized analysis of pictures We are grateful to Dr Jean-Jacques Vasseur for performing MALDI-TOF analysis of 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 and Istituto Superiore di Sanita’, Rome, Italy to M G
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