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- Materials and methods
The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptor-independent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide–lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process. To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.
The pAntp peptide, a 16 amino-acid segment corresponding to the third helix of the homeodomain of Antennapedia protein is known to be internalized by different cell lines [1–4]. Following conjugation, pAntp has been used as a vector for intracellular delivery of molecules such as peptides [5–8] and oligonucleotides [9,10]. The pAntp peptide is taken up into the cytoplasm and the nucleus of cells both at 37 °C and 4 °C indicating that this internalization does not occur by endocytosis . In addition, the reverso, enantio, and retro-inverso forms of pAntp are also imported into cells demonstrating that the internalization is a receptor-independent process [11,12]. Phosphorus NMR studies show that the interaction between pAntp peptide and a dispersion of brain phospholipid induces the lamellar to inverted hexagonal phase transition . It was therefore proposed that the pAntp peptide interacts with phospholipids and translocates through the bilayer by transient formation of inverted micelles in the membrane. This model implies that the peptide remains in an aqueous environment and may explain why hydrophilic compounds linked to the peptide are transported into cells . It has been demonstrated that different amphipathic peptides can translocate through a model membrane either by transient formation of pores in the membrane [15–17] or in potential-dependent manner [18,19]. However, the mechanism by which the peptide translocates across the bilayer remains to be elucidated.
The pAntp peptide adopts an α helical structure in SDS micelles but this structure does not seem important for (pAntp) cellular uptake as analogues in which the helical structure is lost can still translocate into cells . It was previously reported that tryptophanes or the presence of a net positive charge are important for peptide import into cells [1,20] but no systematic studies have been carried out to define the key properties for pAntp translocation activity.
To build up a sequence-activity relationship, we have carried out an Ala-Scan mapping in which each amino-acid of pAntp was replaced by an alanine residue. These substitutions modify the parameters associated with the amphipathic α helix (hydrophobicity and hydrophobic moment). The analogues were fluorescently labeled with an NBD group at the N-terminus in order to assess the relationships between physico-chemical properties and cell association, cellular uptake, lipid-membrane binding and conformation of pAntp peptide.
Flow cytometry was used to measure cellular uptake of pAntp peptide and its analogues in nonadherent human leukemia K562 cells. To accurately measure the fraction of internalized peptide, we used a method based on the selective chemical extinction of the NBD label by dithionite, a membrane-impermeable compound. This reagent was used previously to measure transbilayer distribution of NBD-labeled phospholipids in membrane cells [21,22], in erythrocyte membranes , and to measure the translocation of an amphipathic NBD-labeled peptide through phospholipid vesicles . We have determined the binding affinity of pAntp and its analogues to phospholipid vesicles by generating binding isotherms based on the NBD fluorescence  and studied the conformation of these peptides in a membrane-mimicking environment by circular dichroism spectroscopy.
Materials and methods
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- Materials and methods
Fmoc-PAL-PEG-PS Support Resin and 9-fluorenylmethyloxicarbonyl (Fmoc) amino acids were purchased from Perseptive Biosystems (Hamburg, Germany). Other reagents used for peptide synthesis or NBD labeling included N,N′-diisopropylcarbodiimide (DIPCDI, Fluka), 1-hydroxybenzotriazole (HOBT, Perkin Elmer), N,N-diisopropylethylamine (DIEA, Fluka), dimethylformamide (Perseptive Biosystems) and 4-chloro-7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD-Cl, Fluka). The lipid POPC (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol) were obtained from Avanti Polar Lipids (Alabama, USA). Sodium dithionite, SDS and poly l-lysine were supplied by Sigma (St Louis, MO, USA). Reagents for cell biology include Opti-MEM, RPMI and fetal bovine serum, obtained from Gibco (Cergy-Pontoise, France), and bovine serum albumine (BSA,Pierce).
Peptide synthesis and labeling
All peptides were synthesized according to the Fmoc-tBu strategy using an AMS 422 (ABIMED, Germany). The peptide sequences are reported in Table 1 and helical-wheel projection of pAntp peptide is shown in Fig. 1. Labeling of the N-terminus of the Ala-Scan peptides with the NBD probe was achieved as described elsewhere . Briefly, resin-bound peptide was treated with piperidin [20% (v/v) in dimethylformamide] in order to remove the N-terminal Fmoc protecting group. NBD-Cl was added in dry dimethylformamide (fivefold molar excess) in the presence of DIEA (twofold molar excess) for 6 h under agitation in the dark to selectively label the N-terminal amino group.
Table 1. Sequences of the pAntp peptide and its Ala-Scan analogues.
|pAntp|| RQIKIWFQNRRMKWKK |
|Ap1|| A QIKIWFQNRRMKWKK |
|Ap2|| R A IKIWFQNRRMKWKK |
|Ap3|| RQ A KIWFQNRRMKWKK |
|Ap4|| RQI A IWFQNRRMKWKK |
|Ap5|| RQIK A WFQNRRMKWKK |
|Ap6|| RQIKI A FQNRRMKWKK |
|Ap7|| RQIKIW A QNRRMKWKK |
|Ap8|| RQIKIWF A NRRMKWKK |
|Ap9|| RQIKIWFQ A RRMKWKK |
|Ap10|| RQIKIWFQN A RMKWKK |
|Ap11|| RQIKIWFQNR A MKWKK |
|Ap12|| RQIKIWFQNRR A KWKK |
|Ap13|| RQIKIWFQNRRM A WKK |
|Ap14|| RQIKIWFQNRRMK A KK |
|Ap15|| RQIKIWFQNRRMKW A K |
|Ap16|| RQIKIWFQNRRMKWK A |
Figure 1. Helical wheel representation of pAntp peptide. Hydrophobic and noncharged residues are represented by white squares. Black circles represent basic residues. Residue numbers are indicated.
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The resin was washed with dimethylformamide and treated with deprotecting mixture to cleave the peptides from the resin and deprotect the side-chains. Peptide purification was accomplished by RP-HPLC (Water-prep LC 40, Water) under trifluoroacetic acid 0,01%/acetonitrile gradient condition. Purity as assessed by reverse phase analytic chromatography (Beckman Gold equipped with a Diode Array detector), was over 95% for all peptides by the criterion of relative UV absorbencies at 220 nm and 460 nm. The molecular masses were validated by MALDI-TOF MS (Voyager Elite, Perseptive Biosystems, UK).
K562 cells were cultured in RPMI supplemented with 10% fetal bovine serum. Cells were diluted to 3 × 105 cells per mL one day before the experiment. About 2 × 105 cells per well were plated on 10-well glass coverslip (7 mm diameter) coated with poly l-lysine at a concentration of 0.1% (w/v in water). Cells were preincubated in Opti-MEM at 37 °C for 30 min before incubation with the peptides. Each peptide solution was prepared at a concentration of 20 µm in Opti-MEM by dilution of a stock solution of peptide at 100 µm in phosphate buffer 25 mm (pH 7.5). The cell medium was removed and the peptide solution (40 µL) was added to the cells. After 15 min at 37 °C, the cells were rinsed three times with NaCl/Pi and fixed with a formaldehyde solution (3.7%) containing 1.5% methanol for 10 min at room temperature. The cells were washed three times with NaCl/Pi before being dried and mounted in Mowiol.
The confocal microscope was a Biorad 1024 CLSM system using a Nikon Optiphot II upright microscope and a Argon-Krypton ion laser with excitation line at 488 nm (emission filter: 522/532 nm). A 60 × planachromatic objectif Nikon lens was used. Series of optical sections were collected and projected onto a single image plane in the large sharp 1024 software and processing system at 512 × 512 pixels resolution.
Flow cytometry of cell association of peptide
K562 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum. Cells were diluted at 3 × 105 cells·mL−1 1 day before the experiment. Cell association was measured by flow cytometry using a FACScan (Becton Dickinson). NBD-labeled peptides (1 µm) were incubated with K562 cells (5 × 105 cells·mL−1) in Opti-MEM medium at 37 °C for various periods of time (final volume was 0.5 mL). Thereafter, the cells were washed twice and then resuspended in 0.5 mL of ice-cold NaCl/Pi for flow cytometric analysis. Cell-associated fluorophores were excited at 488 nm and fluorescence was measured at 525 nm. A histogram of fluorescence intensity per cell (1 × 104) was obtained and the calculated mean of this distribution was considered as representative of the amount of cell-associated peptide. A stock solution of dithionite (1 m) was freshly prepared in 1 m Tris solution (pH 10). Following flow cytometric analysis, 5 µL of dithionite stock solution was added to cells maintained at 4 °C and the fluorescence of internalized peptides was measured after 5 min.
For preparation of small unilamellar vesicles (SUVs), a lipid film of desired composition was dried for 3 h and then suspended in buffer [20 mm Tris, 150 mm NaCl and 1 mm EDTA (pH 7.4)] to give a final concentration between 25–30 mm. The suspension was sonicated in ice-cold water for 25 min using a titanium tip ultrasonicator. Titanium and lipid debris were removed by centrifugation at 100 000 g. Dynamic light-scattering measurements (Brookaven Instruments Ltd, USA) confirmed the existence of a single population of vesicles (more than 95% of mass content) with a mean diameter of 31 ± 1 nm. The lipid concentration was determined in duplicate by phosphorus analysis .
Fluorescence measurements were performed in 3 mL quartz cuvettes at 25° ± 0.1 °C under constant magnetic stirring using a SLM AB-2 fluorometer (SLM Instruments, Inc., Urbana, USA). For measuring the binding of NBD-pAntp and its 16 analogues to SUVs, excitation and emission wavelengths were set at 464 nm and 530 nm, respectively. Excitation and emission band-pass were both set at 4 nm. Aliquots of a concentrated stock solution of liposomes (7.5 mm) were added to the peptide solution (0.5 µm). Each measurement was performed after equilibrium was reached. The contribution of scattered light to the observed signal was negligible. The binding isotherms were analyzed as partition equilibrium employing the following formula:
where Xb is defined as the molar ratio of bound peptide per total lipid, Kp corresponds to the partition coefficient, and Cf represents the equilibrium concentration of free peptide in the solution. Knowing the fluorescence intensity as function of the total lipid concentration, the fraction of bound peptide fb can be calculated and it is then possible to calculate Cf, as well as the extent of peptide binding, Xb. In practice, it was assumed that the peptides were initially partitioned only over the outer leaflet of the SUV (60% the total lipid). Therefore, values of Xb were corrected as follows:
The curve resulting from plotting Xb* vs free peptide, Cf, is referred to as the conventional binding peptide isotherm. Surface partition coefficients Kp were calculated from the initial slopes of the conventional binding isotherm (for a detailed description, see ).
Circular dichroism measurement
CD spectra of peptides were obtained on a Jobin Yvon CD6 spectropolarimeter. The spectra were recorded from 185 to 260 nm at room temperature in a quartz optical cell of 0.02 cm path-length. Baseline spectra for SDS were obtained prior to the peptide spectra. The peptides were scanned at a concentration of 75 µm in buffer (150 mm NaF, 20 mm Tris, 0.1 mm EDTA) in the presence of SDS (12.6 mm) to obtain a SDS-to-peptide molar ratio equal to 170. At this ratio, no change in the CD spectra was observed. The helical content was determined from the mean residue ellipticity [Θ] at 222 nm according to the relation [Θ]222 = −30 300[α] − 2340 where [α] is the amount of helix .
Calculation of peptide hydrophobicity and hydrophobic moment
We have calculated for each peptide the helical hydrophobic moment <µH> for the entire sequence on the basis of a perfect helical structure following the formula of Eisenberg . We have calculated the mean residue hydrophobicity <H> for the entire sequence. For calculation of these parameters, we have used the hydrophobic scale based on the octanol/water partition of individual N-acetyl amino-acid residues . This scale correlates well with the hydrophobic scale concerning uncharged amino acid, based on the partition of a set of analogues of an amphipathic peptide from water to a POPC/POPG bilayer .
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- Materials and methods
Our approach to studying the sequence elements important for the pAntp peptide internalization was based on the use of Ala-scan mapping in which each amino acid in the sequence was successively substituted by an alanine. The mutation of the primary sequence of an α helical amphipathic peptide changes its global physico-chemical properties . Labeling peptides with the NBD group allowed the comparison of cellular uptake and phospholipid-binding of pAntp analogues and analysis of the physico-chemical properties required for translocation.
To measure cellular uptake of NBD-labeled pAntp and its analogues, we have used the dithionite method [21,22]. We report here that dithionite treatment of cell-associated NBD-labeled peptide can be used to estimate the peptide fraction localized inside the cell. First, we determined by flow cytometry that after 1 h of incubation with K562 cells at 37 °C and 4 °C, a fraction corresponding, respectively, to about 29% and 13.6% of cell-associated NBD-pAntp was localized intracellularly. The percentage of total internalized peptide was similar to the one reported in another cell line using a different method based on the derivatization of noninternalized peptide by diazotized 2-nitroalanine [3,4]. In addition, our results show that the pAntp peptide internalization in human leukemia K562 cells can occur at 37 °C and to a lesser extent at 4 °C, supporting the view that the internalization mechanism does not involve entirely endocytosis [1,12]. The kinetics of internalization of NBD-pAntp are rapid and reach a plateau after 1 h, corroborating with the results obtained in other cell lines such as lymphocyte cell  and embryonic nerve cells [1,12]. We confirmed by confocal microscopy that indeed the peptide is inside the cell.
In order to determine the key properties necessary for pAntp internalization, we measured cell association and cell uptake of each NBD-pAntp analogue by flow cytometry. The kinetics of cell association of NBD-pAntp was similar to the kinetic of internalization determined by dithionite assay. The amount of cell-associated peptide represents the sum of internalized peptide and of peptide remaining on the cell surface after cells wash. Interestingly, treatment with dithionite reveals that, at equilibrium, the fraction of cell-associated peptide protected from reduction is similar for each analogue. This could mean that the amount of internalized peptide depends on the quantity of peptide initially associated with cell surface, suggesting that the affinity of the peptide for plasma membrane is a determining factor for cell internalization.
Our Ala-scan internalization results show that hydrophobicity of pAntp plays an important role in cellular uptake. For example, substitution of aromatic residues by an alanine diminishes cell internalization. This corroborates previous results showing the importance of Trp residue for pAntp internalization . Mutation of a charged residue (Arg, Lys) within the sequence of pAntp resulted also in a significant decrease in cell uptake. This could explain why analogues of pAntp, in which the first residue (Arg1) or the last residue (Lys16) are not present within the sequence, enter poorly into cells . Our data, concerning the importance of the net charge of the pAntp peptide for its translocation ability, corroborate also those obtained recently by an Ala-Scan mapping of a biotinylated pAntp peptide . However, in these studies, it was reported that internalization of pAntp peptide does not depend significantly on its hydrophobicity . The discrepancy observed with our results could be due to a difference in experimental conditions used such as the cell type, peptide concentration and peptide labeling.
Fluorescence spectra studies suggested that the peptide interacts with the phospholipid matrix of the cellular membrane and supported the involvement of a lipid–peptide interaction during the translocation process . We therefore investigated the role of the sequence in the phospholipid binding of pAntp peptide. Liposomes are lipid bilayer models suitable for determining by fluorescence the influence of substitution on lipid-binding affinity [24,32,34].
We show that both the helical hydrophobic moment and the hydrophobicity modulate the binding of pAntp peptide to the lipid bilayer containing reduced negative surface charge. In addition, the slight diminution of hydrophobicity and helical hydrophobic moment, obtained by substitution of hydrophobic residue, results in a strong decrease of pAntp affinity towards phospholipids. This suggests that pAntp peptide has the minimal helical amphipathicity required to interact with bilayers of low negative surface charge. With an increase in vesicle negative charge, electrostatic interactions between the peptide and the membrane become predominant and compensate for the loss of hydrophobic interactions. Moreover, a statistical analysis indicates that a modification of hydrophobicity or helical hydrophobic moment in the N-terminal half of pAntp peptide could change its affinity for POPC/POPG (75 : 25) SUVs. In contrast, substitutions within the highly charged C-terminal half of the peptide do not influence lipid-binding of peptide probably because electrostatic interactions are predominant and are not influenced by the modification of the hydrophobicity or helical amphipathicity values in this segment. This corroborates a previous study demonstrating that hydrophobic interaction and global helical propensity are essential for the lipid-binding of an amphipathic peptide to a neutral surface but not to a negatively charged bilayer .
The N-terminal half of the pAntp peptide in the presence of SDS is highly ordered in an amphipathic α helix from residues Arg1 to Gln8 . Mutation of Trp6 decreases the helical content and lipid-binding, probably by perturbing the repartition of amino acids between the hydrophilic and hydrophobic sides. This suggests that the hydrophobic side of the helix containing Ile3, Trp6, and Phe7 is important for pAntp binding to the inner part of POPC/POPG membrane. In contrast, mutation of Arg10 or Lys13, which increases the helical amphipathicity, is associated with an increase of the helical content and lipid-binding affinity of pAntp. These mutations should allow the creation of a longer hydrophobic side interacting with the inner part of the phospholipid membrane. This means that the presence of several charged residues does not allow pAntp to adopt a complete amphipathic structure and disturbs its interaction with the phospholipid membrane especially when the charge-density is low.
Although the hydrophobic plot suggests that a minimal value for these both parameters could be necessary for cell uptake of pAntp peptide, no correlation exists between the helical amphipathicity and the amount of internalized peptides. It should be expected, however, that the amount of internalized peptide probably reflects the translocation capacity of the molecule as well as its affinity for cell surface. Interestingly, the modulation of hydrophobicity of pAntp peptide, without modifying its net charge, changes both its affinity for vesicles having low charge density and its uptake into cells. Thus, the interaction of pAntp peptide with cellular surface, if we assume a model in which the peptide interacts with neutral lipid matrix of cell membrane, is probably modulated by hydrophobicity of the peptide. In contrast, no correlation was found between the net charge of pAntp peptide, its lipid-binding affinity and its cellular uptake. In many cases, the substitution of one of the basic residues decreases the internalization of pAntp but increases its lipid-binding affinity. The net charge of the peptide could be important not only for the interaction of pAntp peptide with cell surface but also for others steps of the internalization process.
Taken together, these results indicate that certain physicochemical parameters modulate internalization of pAntp peptide and that a partial relationship exists between the lipid-binding affinity of this peptide and its uptake into cells. Absence of relevant relationship between the cellular uptake and the helical amphipathicity of peptide is in agreement with data obtained recently with a set of α-canonical amphipathic peptides . Indeed, the lack of correlation, between this structure-dependent property and cellular uptake corroborates previous conclusions, which indicate that the helical structure of pAntp is not required for peptide internalization [12,13].
The cellular uptake of the peptide is proportional to its cell association suggesting that the affinity of pAntp peptide for plasma membrane may be a determining factor for its translocation process. To explain its internalization, it was proposed earlier that pAntp peptide translocates into cells by inducing a lamellar to inverted micelle transition in plasma membrane [1,12]. Moreover, it was mentioned that the substitution of tryptophan within the sequence, which abolishes the translocation activity of pAntp, suppresses its membrane-destabilizing properties [13,14]. Our results suggest that the poor cellular uptake observed with several less hydrophobic analogues of pAntp could be explained also by the binding-affinity of peptide to lipid matrix of cellular membranes. The binding isotherm experiment indicates that pAntp association with a phospholipid membrane is a simple phenomenon of aggregation . In addition, we observed that NBD-pAntp peptide and analogues remain localized at the outer leaflet of the vesicle suggesting that no translocation of peptides occurs spontaneously through a lipid membrane. Calcein efflux assay indicates that no permeabilization occurs upon binding of peptide to lipid bilayer (data not shown). This suggests that pAntp, having a minimum helical amphipathicity, interacts with lipid membrane but cannot destabilize the bilayer organization as described for other positively charged amphipathic peptides . This agrees with a recent study suggesting that peptide import cannot be related to destabilization of phospholipid matrix of the plasma membrane but rather to lipid–peptide association which may play a key role in the translocation activity [3,4]. As the pAntp peptide does not seem to translocate through a lipid bilayer, this might indicate that it should interact with other cell surface components to cross cell membrane. It might also be possible that pAntp peptide enters into cells by a mechanism that involves both energy-dependent and independent process as reported for another cell-permeable peptide [3,4]. We are currently extending our studies to better understand the translocation mechanism.