Cellular Internalization of a Cargo Complex with a Novel Peptide

Bioconjugate Chem. 2001, 12, 911−916

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Cellular Internalization of a Cargo Complex with a Novel Peptide Derived from the Third Helix of the Islet-1 Homeodomain. Comparison with the Penetratin Peptide Kalle Kilk,†,§ Mazin Magzoub,‡ Margus Pooga,†,⊥ L. E. Go¨ran Eriksson,‡ U ¨ lo Langel,† and Astrid Gra¨slund*,‡ Department of Neurochemistry and Neurotoxicology, Department of Biochemistry and Biophysics, Arrhenius Laboratories, University of Stockholm, SE-106 91, Stockholm, Sweden; and Department of Biochemistry, University of Tartu, Ravila 19, 50411 Tartu, Estonia; and Estonian Biocenter, Riia 23a, 51010 Tartu, Estonia. Received March 8, 2001

Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide-protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

INTRODUCTION

Homeodomains are approximately 60 amino acids long DNA binding regions of transcription factors (Mu¨ller et al., 1988). It has been shown that several homeodomaincontaining transcription factors exhibit cell-penetrating properties (Chatelin et al., 1996; Prochiantz, A., 1999; Han et al., 2000). For the Antennapedia homeodomain protein of Drosophila, it was found that the fragment corresponding to the third helix (residues 43-58) of the homeodomain penetrates cell membranes (Derossi et al., 1994, 1996, 1998). The peptide with the corresponding sequence was named “penetratin”, and this was the first homeoprotein-derived cell-penetrating peptide (CPP) (Table 1). The cellular internalization of penetratin occurs at 4 °C and is unsaturable, suggesting that it does not require a chiral receptor protein (Derossi et al., 1994). To reveal the mechanism by which penetratin is internalized, modified (with deletions, additions, replacements or inversions) penetratin analogues have been synthesized and studied (Brugidou et al., 1995; Derossi et al., 1996; Fischer et al., 2000; Thore´n et al., 2000; Drin et al., 2001), but a detailed knowledge of the mechanisms of membrane penetration is still lacking. As previous studies demonstrate, the homeodomains have similar and well-organized secondary and tertiary * To whom correspondence should be addressed. Svante Arrhenius va¨g 12. Phone: +46-8-162450. Fax: +46-8155597. E-mail: [email protected]. † Department of Neurochemistry and Neurotoxicology, Stockholm University. ‡ Department of Biochemistry and Biophysics, Stockholm University. § University of Tartu. ⊥ Estonian Biocenter.

structures (Ippel et al., 1999). The homeodomain proteins consist of three R-helices. The third helix contains highly conserved amino acid motifs and is also amphipathic. It has been discussed whether the amphipathic R-helical conformation is essential for the membrane-translocating properties (Dathe et al., 1996 Magzoub et al., 2001). The present study concerns a novel peptide sequence, pIsl, derived from the homeodomain of the rat transcription factor Islet-1 (Isl-1) (Karlsson et al., 1990) and homologous to penetratin. The Islet-1 protein was initially shown to function as an insulin-1 gene enhancer. Later, Isl-1 protein has been shown to possess other important regulatory functions, e.g., in motor neuron differentiation (Pfaff et al., 1996), regulation of amylin (Wang and Drucker, 1996), proglucagon expression (Wang and Drucker, 1995), and estrogen receptor modulation (Gay et al., 2000). The sequence corresponding to the third helix (residues 45-60) of the homeodomain of Isl-1 (pIsl) is similar, although not identical to penetratin (Table 1). The pIsl sequence has only one Trp residue, which is a simplification for studies with optical spectroscopy, and it has a native Cys residue, which may be useful in coupling reactions of cargoes. We demonstrate that pIsl and penetratin peptides internalize efficiently into human Bowes melanoma cells. As shown before, penetratin can translocate relatively large molecules, coupled to it, across the cell membrane (Allinquant et al., 1995; Pooga et al., 1998; Derossi et al. 1998). Here, the biotin-labeled pIsl peptide and penetratin are complexed to fluorescently labeled avidin, a biotin binding protein with a molecular weight of about 63 kDa (Pugliese et al., 1993). The cell translocation of the two complexes is studied and compared. The intrinsic Trp fluorescence in either peptide is used to characterize the interaction of the peptide with negatively charged SDS

10.1021/bc0100298 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/02/2001

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Table 1. Comparison of the Penetratin and pIsl Internalization into Bowes Cells as well as Acrylamide Quenching Constants Calculated from the Tryptophan Fluorescence Spectra in Water and SDS Micelles quenching constant (M-1)

localization name

peptide sequence

cytoplasm

nucleus

water

SDS micelles

penetratin pIsl

RQIKIWFQNRRMKWKK RVIRVWFQNKRCKDKK

+ +

+ +

35 39

25 22

Figure 1. Localization of biotinyl-pIsl (A), biotinyl-penetratin (B), and natural background (C) in Bowes melanoma cells. Concentration of peptides was 10 µM, incubation time 1 h at 4 °C. Staining was done using 150 nM streptavidin-FITC.

micelles, which may be considered as a simple solvent model for a biomembrane. MATERIALS AND METHODS

Peptide Synthesis. The peptides (Table 1) were synthesized using the t-Boc/DCC/HOBt strategy of solidphase peptide synthesis on a PE Applied BioSystems stepwise peptide synthesizer model 431A. The N-terminal biotin was coupled manually using TBTU/HOBt activation, to increase the coupling yield. Cleavage from resin was performed with liquid HF. Highly pure peptides were obtained by purification on a reversed-phase HPLC C18 column. Molecular weights were determined using a plasma desorption time-of-flight mass spectrometer (BioIon) and always corresponded to calculated values. Both peptides were C-terminally amidated. Cells. The human Bowes melanoma cell line was cultivated in a minimal essential medium with Earl-salts (MEM), supplemented with 10% foetal calf serum, 2 mM L-glutamine, 5 IU/mL penicillin, and 5 µg/mL streptomycin, in air enriched with CO2 to 5% at 37 °C. For the penetration assay the cells were grown on round glass coverslips in a 24 well plate to approximately 50% confluence.

Cellular Penetration of Biotin-Labeled Peptides. The serum-containing medium was replaced by a serumfree medium, and an aqueous stock solution of the peptide was added directly into the medium surrounding the cells, to reach the concentration of 10 µM. For detection of a natural level of intracellular biotin and estimation of nonspecific staining, water instead of the peptide solution was added, and in all other respects these negative controls were handled identically to other coverslips. The cells were incubated with peptides for 1 h at 37 °C in 5% CO2 enriched air or 4 °C in air. Thereafter, the cells were washed three times in PBS, fixed, and permeabilized with methanol for 10 min at -20 °C, washed with PBS, and incubated for 1 h in a 5% (w/v) solution of fatfree milk in PBS in order to block nonspecific binding sites. The peptides were visualized by staining with 150 nM streptavidin-FITC (AmershamPharmacia Biotech) for 1 h at room temperature. The cell nuclei were visualized by staining the DNA with Hoechst 33258 (0.5 µg/mL) for 5 min. Thereafter, the coverslips were washed three times in PBS and mounted in 20% glycerol in PBS. The images were obtained by a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss Inc., Germany).

Cell Penetrating Peptides

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Figure 2. Cellular penetration of biotinyl-peptide-avidin-TRITC complexes. Bowes melanoma cells were incubated for 2 h with 5 µM biotinyl-pIsl-avidin-TRITC or biotinyl-penetratin-avidin-TRITC in serum free medium at 37 °C. Nuclei of pIsl treated cells: (A) Hoechst 33258 staining showing DNA, (B) TRITC fluorescence showing avidin transported by pIsl. Nuclei of penetratin treated cells: (C) Hoechst 33258 staining showing DNA, (D) TRITC fluorescence showing avidin transported by penetratin.

Cellular Penetration of Peptide-Cargo Complexes. The cells for protein transport experiments were grown on round glass coverslips in 24-well plates to about 50% confluence. A solution of biotinylated pIsl or penetratin complexed with avidin-TRITC in serum-containing medium was added to the cells. The Bowes cells were incubated for 2 h at 37 °C with 5 µM of peptide-protein complex. The coverslips with cells were washed two times with PBS, fixed with the mixture of 4% paraformaldehyde and 1% glutaric aldehyde in PBS and permeabilized with methanol for 15 min at -20 °C. Nonspecific binding was blocked with 5% BSA in PBS. After final rinsing with PBS, the preparates were mounted in Slow Fade mounting medium (Molecular Probes, Inc.). The localization of the peptide-avidin complexes was examined and photographed by a Vanox AH2-NAS microphotograph system (Olympos) or by a laser confocal scanning microscope MRC-1024 (Bio-Rad). Fluorescence Spectroscopy. Fluorescence spectra were recorded on a Perkin-Elmer LS 50B Luminescence Spectrometer equipped with FL WinLab software. All measurements were made in 4 × 10 mm cuvettes at an ambient temperature of 20 °C. Spectra were recorded in water or a 100 mM solution of sodium dodecyl sulfate (SDS, Kebo), at pH of about 3, well above the critical micellar concentration. The fluorescence was excited at 280 nm, and the emission was recorded from 300 to 500 nm. Emission spectra were recorded with 10 nm excitation and emission bandwidths. For the fluorescence quenching experiments, acrylamide (Sigma) from a 1 M stock solution was added to

the peptide solution, resulting in concentrations between 4 and 100 mM. Quenching constants KSV were determined by a linear regression with the Stern-Volmer equation for a dynamic process (Lakowicz, 1999):

F0 ) 1 + KSV[Q] F

(1)

where F and F0 are the fluorescence intensities in the presence and the absence of acrylamide, respectively, and [Q] is the molar concentration of acrylamide in the solution. RESULTS

Cellular Penetration. The membrane penetration efficacy of pIsl and penetratin peptides was estimated and compared. Bowes melanoma cells were incubated with 10 µM solution of each biotinylated peptide and stained with streptavidin-FITC after fixing the cells. Both peptides yielded identical high intensities of intracellular fluorescence that strongly exceeded the registered natural background (Figure 1). No obvious differences in cellular distribution were observed between pIsl and penetratin, as demonstrated in Figures 1A and 1B. Both peptides were able to penetrate into the cells also at 4 °C (data not shown). The observations show that pIsl, with a sequence analogous to that of penetratin, translocates into the cells in a nonendocytotic manner, because at temperatures below 18 °C endocytosis is abolished. The results also suggest that both peptides penetrate through

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Figure 3. Stern-Volmer plots for the acrylamide quenching of the tryptophan fluorescence from the two homeopeptides in water (open symbols), or 100 mM SDS micelles (filled symbols). The concentration of pIsl (4 2) and penetratin (O b) was 10 µM. Insert: The fluorescence emission spectra (excitation at 280 nm) of pIsl and penetratin in buffer and in 100 mM SDS micelles.

the cell membranes by the same mechanism, and their intracellular localization is similar. Delivery of Cargo Protein over the Plasma Membrane. The property of avidin (MW about 63 kD) to form a very strong and specific complex with biotin was used to demonstrate the ability of pIsl, as well as penetratin, to carry large molecules into the cells. The cell nuclei are shown in Figure 2A and C by staining with the DNAbinding substance Hoechst 33258. The strong complexes between the biotinylated pIsl or penetratin and TRITClabeled avidin were preformed by incubating the components together in a minimal volume for 5 min at room temperature. This solution was added directly into the culture medium over the cells. Incubation of Bowes cells for 30 min or longer with these complexes resulted in accumulation of fluorescent particles in the cells (Figures 2B and 2D), confirming that avidin-TRITC was conveyed into cells also by the pIsl peptide as a noncovalent complex. With both transport peptides, the cargoes are clearly present both in the cytoplasm and in the nucleus of the cells (Table 1). Spectroscopic Studies. Transport peptides have the common feature to interact strongly with biomembrane model systems. The intrinsic Trp fluorescence of the

nonbiotinylated peptides was used to study this interaction. Penetratin contains two Trp residues and pIsl only one (Table 1). Figure 3 (insert) shows the fluorescence emission spectra of penetratin and pIsl in buffer (pH 7) and in 100 mM solution of SDS, a negatively charged detergent that forms micelles in an aqueous solution. In this membrane mimetic solvent both peptides show a 1520 nm (“blue”) shift of the Trp fluorescence emission peak to shorter wavelengths as compared to the aqueous solutions, indicating that the Trp residues interact with a less polar environment than water, i.e., SDS micelles. In a highly hydrophobic environment the wavelength shift of Trp fluorescence could reach 30-35 nm (Lakowicz, 1999). Therefore, the present data suggest a partial shielding from the aqueous solvent. To evaluate the accessibility of Trp residues to the solvent, acrylamide was used as a neutral hydrophilic fluorescence quenching agent. Figure 3 shows the quenching graphs for the two peptides in water and in SDS micelles. In all cases the graphs obey the linear SternVolmer equation. The calculated quenching constants are 35 and 39 M-1 in aqueous solution and of 25 and 22 M-1 in micellar solution, for penetratin and pIsl, respectively (Table 1). The values observed for the two peptides are

Cell Penetrating Peptides

not significantly different within the accuracy of the measurements. It should be noted that the quenching behavior in the current case is also not significantly dependent on the number of Trp residues. DISCUSSION

The present study is the first to demonstrate that a sequence homologous to penetratin taken from a different homeodomain of a transcription factor is also able to carry a large cargo protein through cell membranes. The pIsl peptide efficiently penetrates into the cells in culture and can be therefore considered as a new member of the homeoprotein-derived class of CPPs. We have compared the transport and membrane interaction properties of two homeodomain-derived peptides, penetratin and pIsl, which have somewhat different sequences and net charges (Table 1), but with identical positions of the positive charges. Recently, it was demonstrated that only the C-terminus of penetratin, RRMKWKK, is obligatory for cellular penetration (Fischer et al., 2000). Corresponding sequences are present in many of the known homeoproteins. In the transcription factor PDX-1 the same motif is present, and it was demonstrated to be a nuclear localization signal (NLS) for proteins (Moede et al., 1999). However, in that study the tagged cargo was introduced into the cells by a transfection technique. The results suggest that the translocation property of the CPPs is mainly dependent on the presence of the positive charges in the C terminus. Moreover, L-Alascanning showed that all basic amino acid residues in the RRMKWKK fragment are necessary for efficient translocation (Fischer et al., 2000). Substitution of any charged residue in this motif by L-Ala decreases the penetration yield by 60% or more, whereas replacing Trp does not cause any such drastic drop in the penetration yield. Our results support the previous observations on penetratin analogues that the Trp56 of penetratin is not a major determinant for cellular penetration (Fischer et al., 2000). In a solution with SDS micelles the single Trp of pIsl shows similar fluorescence properties as the two Trp residues in penetratin do. These results suggest that both Trp residues in penetratin are in an environment of similar polarity and accessiblity from the solvent as the one Trp residue in pIsl. In both peptides, the Trp residues are partially shielded from the interaction with the aqueous solvent. The detailed secondary structures induced in a cellpenetrating and transporting peptide by the interaction with a membrane do not seem to be directly related to the penetration properties (Derossi et al., 1996; Scheller et al., 2000; Drin et al. 2001; Magzoub et al., 2001). Instead, the strength of the interaction with the charged membrane and possibly the overall topology of the peptide relative to the membrane surface may be decisive factors for the translocation process. How the cargo will then then become accommodated in a nonlytic manner in the translocation process remains a challenging phenomenon. The presentation of the pIsl peptide as a novel CPP and comparing its properties with penetratin gives further understanding of the cell-penetrating mechanisms of CPPs. Additionally, the native Cys residue in the pIsl sequence may be used for various coupling reactions, which should make pIsl useful as an alternative for cellular delivery of different hydrophilic cargo molecules.

Bioconjugate Chem., Vol. 12, No. 6, 2001 915 ACKNOWLEDGMENT

This study was supported by grants from the Swedish Natural Science Research Council, the Swedish Technological Research Council, the EU programs contract nos. MAS3-CT97-0156 and BIO4-98-0227, the Swedish Institute Visby Program, and the Estonian Science Foundation (ESF 4007). LITERATURE CITED (1) Allinquant, B., Hantraye, P., Mailleux, P., Moya, K., Bouillot, C., and Prochiantz, A. (1995) Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro. J. Cell Biol. 128, 919-927. (2) Berlose, J. P., Convert, O., Derossi, D., Brunissen, A., and Chassaing, G. (1996) Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane-mimetic environments. Eur. J. Biochem. 242, 372-386. (3) Brugidou, J., Legrand, C., Mery, J., and Rabie, A. (1995) The retro-inverso form of a homeobox-derived short peptide is rapidly internalised by cultured neurons: a new basis for an efficient intracellular delivery system. Biochem. Biophys. Res. Commun. 214, 685-693. (4) Chatelin, L., Volovitch, M., Joliot, A. H., Perez, F., and Prochiantz, A. (1996) Transcription factor hoxa-5 is taken up by cells in culture and conveyed to their nuclei. Mech. Dev. 55, 111-117. (5) Dathe, M., Schumann, M., Wieprecht, T., Winkler, A., Beyermann, M., Krause, E., Matsuzaki, K., Murase, O., and Bienert, M. (1996) Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 35, 12612-12622. (6) Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444-10450. (7) Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptorindependent. J. Biol. Chem. 271, 18188-18193. (8) Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8, 84-87. (9) Drin, G., De´me´ne´, H., Temsamani, J., and Brasseur, R. (2001) Translocation of the pAntp peptide and its amphipathic analogue AP-2AL. Biochemistry 40, 1824-1834. (10) Fischer, P. M., Zhelev, N. Z., Wang, S., Melville, J. E., Fahraeus, R., and Lane, D. P. (2000) Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin. J. Pept. Res. 55, 163172. (11) Gay, F., Anglade, I., Gong, Z., and Salbert, G. (2000) The LIM/homeodomain protein islet-1 modulates estrogen receptor functions. Mol. Endocrinol. 14, 1627-1648. (12) Han, K., Jeon, M.-J., Kim, K.-A., Park, J., and Choi, S. Y. (2000) Efficient intracellular delivery of GFP by homeodomains of Drosophila fushi-tarazu and engrailed proteins. Mol. Cells 10, 728-732. (13) Ippel, H., Larsson, G., Behravan, G., Zdunek, J., Lundqvist, M., Schleucher, J., Lycksell, P. O., and Wijmenga, S. (1999) The solution structure of the homeodomain of the rat insulingene enhancer protein isl-1. Comparison with other homeodomains. J. Mol. Biol. 288, 689-703. (14) Joliot, A. H., Triller, A., Volovitch, M., Pernelle, C., and Prochiantz, A. (1991) alpha-2,8-Polysialic acid is the neuronal surface receptor of Antennapedia homeobox peptide. New Biol. 3, 1121-1134. (15) Karlsson, O., Thor, S., Norberg, T., Ohlsson, H., and Edlund, T. (1990) Insulin gene enhancer binding protein Isl-1 is a member of a novel class of proteins containing both a homeo- and a Cys-His domain. Nature 344, 879-882.

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