Physico-chemical requirements for cellular uptake of pAntp peptide - Role of lipid-binding affinity

The pAntp peptide, corresponding to the third helix of the Antennapedia hom eodomain, 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-Sca n 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 flo w cytometry. Furthermore, we determined the fraction of internalized peptid e 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 physi co-chemical requirements for phospholipid-binding of pAntp, we have determi ned the surface partition coefficient of peptides by titrating them with ph ospholipid vesicles having different compositions. In addition, we estimate d by circular dichroism the conformation adopted by these peptides in a mem brane-mimetic environment. We show that the phospholipid binding of pAntp d epends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, r elated to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phosph olipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mecha nism.