Pathway for polyarginine entry into mammalian cells

Abstract

Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R(9)), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R(9) 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 R(9) 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 R(9) for heparin (K(d) = 109 nM). Finally, R(9) 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.

Figure 1

Localization of TAMRA–R₉ in living and fixed cells. The internalization of TAMRA–R₉ (1.0 μM) was analyzed by fluorescence microscopy. Cells were counterstained with Hoescht 33342. (A) living CHO-K1 cells. (B) CHO-K1 cells fixed with paraformaldehyde. Bar, 10 μm.

Figure 2

Co-localization of TAMRA–R₉ in living cells. The internalization of TAMRA–R₉ (1.0 μM) and marker dyes into living CHO-K1 cells was analyzed by fluorescence microscopy. (A) TAMRA–R₉. (B) FM 1-43. (C) TAMRA–R₉, FM 1-43, and Hoescht 33342. (D) Bright-field image of CHO-K1 cells. Bar, 10 μm.

Figure 3

Dependence of TAMRA–R₉ internalization into living cells on glycosaminoglycans. Internalization of TAMRA–R₉ (1.0 μM) into living wild-type and mutant CHO cells. (A) CHO-K1 cells (wild-type). (B) CHO–pgsD-677 cells (which lack HS). C, CHO–pgsA-745 cells (which lack HS and CS). Bar, 10 μm.

Figure 4

Affinity of TAMRA–R₉ for heparin. (A) Prevalent structural motif in heparin. (B) Elution profile from affinity chromatography of TAMRA–R₉ on immobilized heparin. TAMRA–R₉ was eluted with a linear gradient of NaCl (0–2 M) (conductivity, thin line), and monitored by its absorbance at 280 nm (thick line). (C) Fluorescence titration of TAMRA–R₉ (1.0 nM) with heparin (M_r 3000) in 20 mM HEPES–NaOH buffer, pH 7.5. The equilibrium dissociation constant is K_d = 109 ± 13 nM.

Figure 5

R₉-Induced leakage of egg PC liposomes. (A) Time course for the leakage of carboxyfluorescein from egg PC liposomes (100-μm diameter) upon exposure to various concentrations of R₉. (B) Leakage of carboxyfluorescein from egg PC liposomes (100-μm diameter) as a function of peptide:lipid molar ratio. Data in panel B are derived from data in panel A at 180 s.

Figure 6

Pathway for the transduction of R₉ into cells. Cationic PTDs such as R₉ bind to HSPGs on the cell surface. PTDs are internalization by endocytosis. HS is degraded by heparanases. Free PTDs leak from endocytic vesicles and enter the cytosol.