Efficiency of Protein Transduction Is Cell Type-dependent and Is Enhanced by Dextran Sulfate

Abstract

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly utilized to deliver biologically active agents to a variety of cell types in vitro and in vivo. We report that in addition to previously characterized arginine-rich PTDs, including TAT, lysine homopolymers were able to mediate transduction of a wide variety of cell types, as measured by flow cytometric and enzymatic assays. The efficiency of PTD-mediated transduction was influenced by the cell type tested, although polylysine homopolymers demonstrate levels of internalization that consistently exceeded those of TAT and arginine homopolymers. Transduction of arginine/lysine-rich PTDs occurred at 4°C and following depletion of cellular ATP pools, albeit generally at reduced levels. Although transduction was reduced in Chinese hamster ovary mutant lines deficient in either heparan sulfate or glycosaminoglycan synthesis, uptake was restored to wild-type levels by incubating target cells with dextran sulfate. The enhancement of transduction by dextran sulfate suggests that electrostatic interactions play an important first step in the process by which PTDs and their cargoes traverse the plasma membrane.

Pathway for Polyarginine Entry into Mammalian Cells

“…Early mechanistic studies on the cellular entry of PTDs were performed with fixed cells (11,13,(22)(23)(24)(25)(26)(27)(28). Fixation is believed to permeate membranes (30), and allows vesicle-entrapped peptides and proteins to travel to new locations (29,30,32).…”

“…Internalization was found to be energy-dependent, and the internalized peptide was observed in the endocytic vesicles of living cells (30). Dervan, Johansson, and their coworkers have reported similar cell-fixation artifacts for the transduction of N-methylpyrrole/Nmethylimidazole polyamides (31) and PTD-green fluorescent protein fusions (32).There have also been conflicting reports on whether PTDs bind to cell-surface glycosaminoglycans (GAGs) (11,27,33,34). The cell surface is coated with polysaccharides such as HS and sialic acid, and these molecules are known to mediate the binding of many proteins to cells (34).…”

“…In the presence of a lipid bilayer, penetratin appears to form an amphipathic α-helix, which facilitates its insertion into the bilayer (44,45). 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 (11,13,(22)(23)(24)(25)(26)(27)(28). Fixation is believed to permeate membranes (30), and allows vesicle-entrapped peptides and proteins to travel to new locations (29,30,32).…”

Enhancement of HIV-1 Tat fusion protein transduction efficiency by bog blueberry anthocyanins

“…Protein transduction technology based on small domains called protein transduction domains (PTDs) and cell penetrating peptides (CPPs) has been used to deliver exogenous protein into cultured cells and animal models (2)(3)(4)(5)(6)(7). Many research groups have demonstrated the efficacy of protein transduction technology for protein therapy strategies as well as drug delivery, although the exact mechanism of transduction remains unclear (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Although various proteins have been developed for protein therapy, protein transduction technology has several limitations such as transduction efficiency.…”

Human cytomegalovirus protein pp65: an efficient protein carrier system into human dendritic cells

“…[22][23][24][25][26] However, the PTD-mediated uptake, which is initiated by ionic interactions with the cell surface, still requires high protein concentrations. 24,[27][28][29][30][31] As this limits the stimulation of T cells by PTD-coupled proteins in vivo, more efficient delivery systems are desirable.…”