Pathway for polyarginine entry into mammalian cells,Biochemistry 43

ABSTRACT: Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R9), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R9 is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Colocalization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R9 to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R9 for heparin (Kd) 109 nM). Finally, R9 is shown to promote the leakage of liposomes but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves (1) binding to cell surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles. Facilitating the delivery of molecules into mammalian cells is of substantial interest (1-6). Developing the means to do so can generate both insights into cellular function and potential applications in biomedicine. As early as 1965, Ryser noted that mixing polylysine or other cationic macromol-ecules with proteins greatly increases the uptake of those proteins by cells (7). In 1988, Frankel and Green indepen-dently discovered that the RNA-binding HIV1 TAT protein was capable of crossing lipid bilayers (8, 9). The dissection of HIV TAT function has revealed that a small cationic domain, residues 47-57, is responsible for transduction (10). Other cationic, nucleic acid-binding sequences are likewise capable of entering cells (11, 12). Peptide-like molecules and even nonpeptidyl polycations are capable of crossing lipid bilayers (11, 13). These cationic “protein transduction domains ” (PTDs) not only are capable of entering cells but also can be used to deliver molecular cargo (14-16), such as proteins, oligonucleotides, and small molecules (1, 17