No abstract

Cell-penetrating peptides (CPPs) have been extensively studied during the past decade, because of their ability to promote the cellular uptake of various cargo molecules, e.g., oligonucleotides and proteins. In a recent study of the uptake of several analogues of penetratin, Tat(48-60) and oligoarginine in live (unfixed) cells [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107], it was found that both endocytotic and nonendocytotic uptake pathways are involved in the internalization of these CPPs. In the present study, the membrane interactions of some of these novel peptides, all containing a tryptophan residue to facilitate spectroscopic studies, are investigated. The peptides exhibit a strong affinity for large unilamellar vesicles (LUVs) containing zwitterionic and anionic lipids, with binding constants decreasing in the order penetratin > R(7)W > TatP59W > TatLysP59W. Quenching studies using the aqueous quencher acrylamide and brominated lipids indicate that the tryptophan residues of the peptides are buried to a similar extent into the membrane, with an average insertion depth of approximately 10-11 A from the bilayer center. The membrane topology of the peptides was investigated using an assay based on resonance energy transfer between tryptophan and a fluorescently labeled lysophospholipid, lysoMC, distributed asymmetrically in the membranes of LUVs. By determination of the energy transfer efficiency when peptide was added to vesicles with lysoMC present exclusively in the inner leaflet, it was shown that none of the peptides investigated is able to translocate across the lipid membranes of LUVs. By contrast, confocal laser scanning microscopy studies on carboxyfluorescein-labeled peptides showed that all of the peptides rapidly traverse the membranes of giant unilamellar vesicles (GUVs). The choice of model system is thus crucial for the conclusions about the ability of CPPs to translocate across lipid membranes. Under the conditions used in the present study, peptide-lipid interactions alone cannot explain the different cellular uptake characteristics exhibited by these peptides.

The potential use of polypeptides and oligonucleotides for therapeutical purposes has been questioned because of their inherently poor cellular uptake. However, the 16-mer oligopeptide penetratin, derived from the homeodomain of Antennapedia, has been reported to enter cells readily via a non-endocytotic and receptor-and transporter-independent pathway, even when conjugated to large hydrophilic molecules. We here present the first study where penetratin is shown to traverse a pure lipid bilayer. The results support the idea that the uptake mechanism involves only the interaction of the peptide with the membrane lipids. Furthermore, we conclude that the translocation does not involve pore formation. ß

The binding of penetratin, a peptide that has been found useful for cellular delivery of large hydrophilic molecules, to negatively charged vesicles was investigated. The surface charge density of the vesicles was varied by mixing zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) at various molar ratios. The extent of membrane association was quantified from tryptophan emission spectra recorded during titration of peptide solution with liposomes. A singular value decomposition of the spectral data demonstrated unambiguously that two species, assigned as peptide free in solution and membrane-bound peptide, respectively, account for the spectral data of the titration series. Binding isotherms were then constructed by least-squares projection of the titration spectra on reference spectra of free and membrane-bound peptide. A model based on the Gouy-Chapman theory in combination with a two-state surface partition equilibrium, separating the electrostatic and the hydrophobic contributions to the binding free energy, was found to be in excellent agreement with the experimental data. Using this model, a surface partition constant of approximately 80 M(-)(1) was obtained for the nonelectrostatic contribution to the binding of penetratin irrespective of the fraction of negatively charged lipids in the membrane, indicating that the hydrophobic interactions are independent of the surface charge density. In accordance with this, circular dichroism measurements showed that the secondary structure of membrane-associated penetratin is independent of the DOPC/DOPG ratio. Experiments using vesicles with entrapped carboxyfluorescein showed that penetratin does not form membrane pores. Studies of the cationic peptide penetratin are complicated by extensive adsorption to surfaces of quartz and plastics. By modification of the quartz cell walls with the cationic polymer poly(ethylenimine), the peptide adsorption was reduced to a tolerable level. The data analysis method used for construction of the binding isotherms eliminated errors emanating from the remaining peptide adsorption, which otherwise would prevent a proper quantification of the binding.

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from K K-helix to antiparallel L L-pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.