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Cellular translocation of proteins by transportan¹

MARGUS POOGA^*,, CECILIA KUT^*,, MADELEINE KIHLMARK^,, MATTIAS HÄLLBRINK^*, SANDRA FERNAEUS^*, RAIVO RAID^¶, TIIT LAND^*, EINAR HALLBERG, TAMAS BARTFAI and ÜLO LANGEL^*,2

^* Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden;
Estonian Biocentre, EE-51010, Tartu, Estonia;
Section for Natural Sciences, Södertörns Högskola (University College), S-141 04 Huddinge, Sweden;
Department of Biochemistry, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden;
^¶ Institute of Zoology and Hydrobiology, Tartu University, EE-51014, Tartu, Estonia; and the
Harold L. Dorris Neurological Research Center, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA

²Correspondence: Harold L. Dorris Neurological Research Ctr., Department of Neuropharmacology, The Scripps Research Inst.,10550 N. Torrey Pines Rd., Mail SR-307, La Jolla, CA 92037, USA. E-mail: ulangel{at}scripps.edu

SPECIFIC AIMS

Peptide-mediated delivery of hydrophilic macromolecules has gained more and more attention in recent years. Transduction of protein of interest into cells by the help of peptides can serve as a good alternative to protein microinjection and protein expression from plasmids in transfected cells.

In this study, we investigated whether a small cell-penetrating peptide transportan is able to deliver whole intact proteins across the plasma membrane into the cell interior. We also studied the possibility of applying peptide-mediated transport for noncovalent complexes and confirmed that unfolding of proteins is not obligatory for delivery into cells.

PRINCIPAL FINDINGS

1. Transportan is able to deliver medium-sized proteins into cells
The green fluorescent protein (GFP), a widely exploited tag, was used to estimate the ability of transportan to deliver intact proteins into cells. We coupled recombinant GFP to transportan (TP) via a disulfide bridge formed between an extra cysteine at the GFP carboxyl terminus and Cys--NH-Lys¹³ of TP (Fig. 1A ). The transportan-GFP conjugate was added directly to the cell culture medium, and after 10–60 min the GFP fluorescence was located in the interior of the cells (Fig. 2 ). Most of the GFP fluorescence was located to cytoplasmic membranes and to a lesser extent to the plasma membrane. The intensity and localization of GFP fluorescence in the plasma membrane were not affected by treatment of cells with reducing agents, suggesting that the disulfide bond of the construct and the construct itself are extracellularly inaccessible, and hence must be located inside the cells or buried in the membrane.

View larger version (21K): [in this window] [in a new window]   Figure 1. Different strategies for attaching transportan to the cargo protein by the labile disulfide bond (A), by forming a noncovalent complex (B), or by using a bifunctional chemical cross-linker (C).

View larger version (79K): [in this window] [in a new window]   Figure 2. Cellular uptake of the GFP-transportan conjugate. BRL or COS-7 cells on glass coverslips were incubated with GFP-transportan conjugate at 37°C in serum-free medium. Corresponding images of GFP fluorescence (A), DNA staining (B), and phase contrast (C) through the mid plane of BRL cells incubated with 0.5 µM GFP-transportan for 10 min. GFP fluorescence in sections close to the coverslip surface of BRL cells were incubated with 0.2 µM GFP-transportan for 1 h (D, E). Cells depicted in panel E were washed with buffer containing 1 mM DTT before fixation. Confocal images of GFP fluorescence from a 0.3 µm section below (F) or through (G) the nuclear equator of COS-7 cells and a 0.3 µm section close to the surface of the coverslip of another COS-7 cell (H) incubated with 0.2 µM GFP-transportan for 1 h. The GFP fluorescence distributed mainly in a reticular pattern in the cytoplasm (large arrowheads in panels A, F) and in the plasma membrane and boundaries between the cells (small arrowheads in D, E, H), but was absent form the nucleoplasm or cytoplasm (arrows in panel G). Bars 10 µm.

2. GFP is internalized in an intact folded state
Native GFP has a very tight barrel-shaped structure that has to be preserved for fluorescence. The fluorescence is detectable at all stages of TP-GFP internalization: in the solution, at the plasma membrane, and inside the cells (Fig. 2) , suggesting that GFP in the TP-GFP construct does not unfold during passage over the plasma membrane.

3. Large proteins coupled to transportan are delivered into cells
Not only medium-sized conjugates of transportan with proteins (e.g., GFP-TP) are able to translocate into cells. Coupling transportan to avidin-TRITC conjugate by a chemical cross-link results in a cell-translocating protein derivative (Fig. 1C ). Cellular uptake of transportan-avidin-TRITC construct was rapid: 5 min incubation with the construct led to accumulation of the label in the plasma membrane of COS-7 cells. After 30 min, avidin was detectable inside the cells. Coupling of transportan molecules to protein gives cell-translocating ability to as large proteins as antibodies.

4. The covalent bond between the transport peptide and a cargo is not necessary for protein delivery
The efficient delivery of a cargo molecule into the cells is considered to be achieved only when coupled covalently to the transport peptide. On the other hand, the possibility of using a noncovalent complex of the transport peptide and a cargo molecule for delivery has not been much studied so far.

A fluorescent derivative of streptavidin (e.g., streptavidin-Texas red) itself or in a combination with a cell-penetrating peptide is not able to translocate from the tissue culture medium into the cell interior (Fig. 3E ). Simultaneous addition of biotinylated transportan and Texas red-labeled streptavidin (Fig. 1) leads to the formation of strong complexes and to the insertion of complexes into the plasma membrane of COS-7 cells in 5–10 min. Later, these complexes shift more inside the cells and finally concentrate in the perinuclear area (Fig. 3A , B , C , D ).

View larger version (84K): [in this window] [in a new window]   Figure 3. Delivery of streptavidin-Texas red conjugate (SA-TxR) by biotinyl-transportan into COS-7 cells. Consecutive images of one cell with a distance of 1 µm between the planes (A–D). The cells were incubated for 2 h at 37°C with 2 µM biotinyl-transportan and streptavidin-Texas red (red) (1:100 dilution) in IMDM. Nonbiotinylated transportan was applied into the culture medium along with SA-TxR (E). The plasma membrane was visualized with concanavalin A-FITC (green) (2 µg/ml). Uptake of biotinyl-TP-SA-TxR at 0°C (2 h incubation) (F). Uptake of biotinyl-TP-SA-gold complexes by Bowes melanoma cells. The cells were incubated for 2 h at 37°C with 2 µM biotinyl-TP and SA-gold (diameter 10 nm, 1:50 dilution) (G). Delivery of anti-biotin monoclonal antibody into Bowes cells by biotinyl-transportan. Cells were incubated with 2 µM biotinyl-TP and 1 µg/ml anti-biotin antibody (clone 33 Boehringer Mannheim) in culture medium for 2 h at 37°C and stained after fixation with anti-mouse-FITC (Sigma 1:100) (H).

Preformation of the complex between biotinyl-transportan and streptavidin-TxR before applying to the cells for 5–15 min slightly increased the initial velocity of the uptake, but the general pattern of localization and distribution of complexes inside the cells remains unchanged.

The delivered streptavidin is confined to vesicular structures in COS-7 cells (Fig. 3A , B , C , D ). Complexes of biotinyl-transportan with streptavidin-gold conjugate (SA-Au) reside in vesicular structures in COS-7 cells that are either surrounded by membrane or not, but are also diffuse in the cytoplasm, as judged by electron microscopy (Fig. 3G ). Localization of delivered SA-Au outside the membrane surrounded vesicles suggests that peptide-mediated uptake of proteins can also involve processes other than absorptive endocytosis. Moreover, lowering temperature below 18°C abolishes endocytosis, but the uptake of biotinyl-transportan complexes with streptavidin-Texas red is not completely suppressed even at 0–4°C (Fig. 3F ).

5. Transportan is able to deliver large complexes into the cell interior
Avidin and streptavidin form extremely strong complexes with biotin and biotinylated molecules. Labeled streptavidin was efficiently delivered into the cells by biotinylated transportan, most probably in the form of a complex. It is still unclear how stable the complexes of a transport peptide with a protein have to be and how large these proteins can be in order to be efficiently translocated into cells. Monoclonal anti-biotin antibodies with a molecular mass 150 kDa are also conveyed into the cells by biotinylated transportan (Fig. 3H ), demonstrating that the complex of peptide and protein does not have to be extra strong. The size of the proteins to be delivered intracellularly by transportan probably is not limited to a molecular mass of 150 kDa. The particle size of streptavidin conjugate with colloidal gold corresponds to a globular protein with a molecular mass of 1 MDa; in a complex with biotinyl-transportan, this conjugate translocates efficiently in a noninvasive way into living cells in culture.

CONCLUSIONS

Cell-penetrating peptides have been used to noninvasively transport small cargoes like oligonucleotides and peptides into the living cells for 4–5 years. Recently, peptide-mediated cellular delivery of whole proteins was demonstrated. Transportan along with Tat and Antennapedia-derived peptides conveys proteins into living cells.

How these cell-penetrating peptides or protein-transducing domains enter the cell is largely unknown. Their internalization is not mediated by endocytosis, chiral receptors, or proteins in general and is not dependent on temperature or ATP. There is only one model (formation of inverted micelle) proposed for penetration of Antennapedia-derived peptide into cell, but it has not yet been proved experimentally.

Even less is known about the mechanism of peptide-mediated protein transport. Formation of pores in the plasma membrane of a cell by penetrating peptides, thereby enabling direct access to cell interior for proteins, can be excluded since mixing of transport peptide with a cargo protein does not lead to protein translocation into the cells. Moreover, formation of even short-living pores by cell-penetrating peptides has never been detected.

Proteins that are coupled to transport peptide by a chemical bond are efficiently delivered cellularly. However, the covalent bond between a cargo protein and the penetrating peptide is not necessary, since the cells also internalize noncovalent transportan–protein complexes. Very large complexes/conjugates that correspond to 1 MDa protein can be conveyed into living cells by transportan. The peptide–protein complexes are not internalized by the cells by endocytosis alone; other, less understood mechanisms must be involved: the complexes translocate into cells at low temperatures and a significant fraction of the protein delivered inside the cells is localized outside the membrane-surrounded vesicles.

Some peptides enable transport of large proteins and protein complexes into cells in a noninvasive way, which can be used to regulate the finely tuned cellular processes. Unfolding of protein by denaturation is not necessary for cellular delivery; inside the cells, at least a fraction of transported protein is located outside the membrane surrounded vesicles and is not targeted into lysosomes. Accordingly, transportan enables transduction of whole proteins into the cells, where they retain their intactness and activity (at least partially).

Our data suggest that transportan is able to deliver proteins and other hydrophilic macromolecules into mammalian cells and demonstrate its potential as powerful cellular delivery vector for scientific and therapeutic purposes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0780fje ; to cite this article, use FASEB J. (April 18, 2001) 10.1096/fj.00-0780fje

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