The efficacy of nascent chemotherapeutics can be limited by their ability to enter mammalian cells (43
). PTDs can expedite delivery (1
). Determining the requirements for PTD entry into cells could enable the creation of even better molecular devices to effect internalization, as well as reveal new aspects of cellular biochemistry and biophysics.
Previous work has led to conflicting conclusions regarding the interaction of PTDs with cells. The mechanism by which peptide toxins, such as mellitin and magainin, interact with cells has been studied extensively, and these peptides are known to form pores in the plasma membrane (42
). Penetratin, which is a cationic peptide corresponding to the third α-helix of the Antennapedia homeodomain, is capable of crossing membranes without causing vesicle disruption. In the presence of a lipid bilayer, penetratin appears to form an amphipathic α-helix, which facilitates its insertion into the bilayer (44
). HIV TAT peptide has likewise be found in a helical structure by NMR spectroscopy, suggesting that HIV TAT and perhaps other PTDs could act in a manner similar to penetratin (46
Early mechanistic studies on the cellular entry of PTDs were performed with fixed cells (11
). Fixation is believed to permeate membranes (30
), and allows vesicle-entrapped peptides and proteins to travel to new locations (29
). Likewise, we find that TAMRA–R9
is found in different cellular locations in fixed and living cells. Specifically, TAMRA–R9
localizes in the nucleolus and cytoplasm of fixed cells (). In living cells, however, TAMRA–R9
is found primarily in vesicles ( and ). As a result, our efforts to elucidate the requirements for PTD internalization have focused on living cells.
In living cells, cationic proteins such as HIV TAT protein and some analogues of ribonuclease A (which is a small cationic protein (47
)) are thought to bind to anionic carbohydrates on the cell surface (33
). Moreover, polysaccharides such as heparan sulfate are believed to play important roles in cellular signaling by initiating the binding of certain proteins to their cellular receptors (34
). On the cell surface, heparan sulfate is attached to either transmembrane proteins to form syndecans or GPI-anchored proteins to form glypicans (51
). The HS side chains of these heparan sulfate proteoglycans (HSPGs) undergo hydrolytic degradation by heparanases en route
to the lysosome (53
A number of proteins are known to bind to specific HSPGs. For some of these proteins, HS is believed to act as the receptor for cellular entry (54
). We find that the entry of a cationic PTD into living mammalian cells relies on the presence of HSPGs. Specifically, the entry of TAMRA–R9
into CHO cells that are deficient in HS is decreased greatly relative to that into wild-type cells (). Similar results have been observed with the intact HIV-1 TAT protein (54
HS and heparin are analogous glycosaminoglycans (51
Accordingly, heparin has been used as a surrogate for HS in a wide variety of biochemical experiments (56
). We reasoned that if HSPGs mediate the cellular entry of PTDs, then cationic peptides such as TAMRA–R9
should have a measurable affinity for heparin. Indeed, we find that TAMRA–R9
binds strongly to immobilized heparin (), which mimics the HS side chains of cell-surface HSPGs. Moreover, the soluble heparin·TAMRA–R9 complex has a value of Kd
near 0.1 μM (), which is similar to that of a typical receptor–ligand interaction.
How does R9 release from HS and escape from endocytic vesicles? HS is cleaved by heparanases, first in vesicles of neutral pH and then in acidic endosomes (34
). HS cleavage would diminish its anion valency and should thus diminish its affinity for R9. We find that free R9
is capable of disrupting a lipid bilayer and causing a liposome to leak (). This leakage is maximal between pH 7.5 and 9.5 (data not shown), suggesting that R9
could escape from endosomes prior to their acidification. Regardless, R9
-induced liposomal leakage occurs at a peptide:lipid molar ratio that is 10- to 100-fold greater than that for known pore-forming peptides (38
). Thus, the disruption of lipid bilayers is likely to play a limited role in transduction at low concentrations of R9
, and a relatively high concentration could be necessary for efficient transduction.
Our findings are consistent with a four-step pathway for the entry of cationic PTDs into the cytoplasm of mammalian cells (). First, peptides bind to HSPGs on the cell surface. These peptides are then taken up a cell by heparan sulfate-mediated endocytosis. Once in vesicles, HS is degraded by heparanases, releasing the PTD. Finally, unbound PTD escapes from the vesicles after achieving a concentration high enough to promote vesicular leakage. This pathway bears some analogy to those proposed for the cellular entry of other HS-binding proteins, such as bFGF (60
) or follistatin (61
). The identity of the rate-limiting step for PTD entry () is not known. Identifying that step could lead to more efficacious PTDs or to transduction agonists.
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.