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  ne  tique Mole  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  ne  tique Mole  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: vives@igm.cnrs-mop.fr 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. 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