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- Materials and Methods
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To understand the protein transduction domain (PTD)-mediated protein transduction behavior and to explore its potential in delivering biopharmaceutic drugs, we prepared four TAT–EGFP conjugates: TAT(+)–EGFP, TAT(−)–EGFP, EGFP–TAT(+) and EGFP–TAT(−), where TAT(+) and TAT(−) represent the original and the reversed TAT sequence, respectively. These four TAT–EGFP conjugates were incubated with HeLa and PC12 cells for in vitro study as well as injected intraperitoneally to mice for in vivo study. Flow cytometric results showed that four TAT–EGFP conjugates were able to traverse HeLa and PC12 cells with almost equal transduction efficiency. The in vivo study showed that the TAT–EGFP conjugates could be delivered into different organs of mice with different transduction capabilities. Bioinformatic analyses and CD spectroscopic data revealed that the TAT peptide has no defined secondary structure, and conjugating the TAT peptide to the EGFP cargo protein would not alter the native structure and the function of the EGFP protein. These results conclude that the sequence orientation, the spatial structure, and the relative location of the TAT peptide have much less effect on the TAT-mediated protein transduction. Thus, the TAT-fused conjugates could be constructed in more convenient and flexible formats for a wide range of biopharmaceutical applications.
Most of the biopharmaceutical molecules, such as peptides, proteins, enzymes, and oligonucleotides, have limited therapeutic effectiveness because they lack the intrinsic ability to traverse the plasma membrane. A number of short peptides called protein transduction domains (PTDs) or cell-penetrating peptides (CPPs) have demonstrated the ability to cross the plasma membranes effectively (1–5). The most extensively studied PTDs are from HIV-1 transactivator of transcription protein (TAT), drosophila homeodomain transcription factor antennapedia (ANTP), and herpes simplex virus structural protein VP22 (6–8). These peptides are rich in arginine or lysine, and it is proposed that interactions between the positively charged PTDs and the negatively charged proteoglycans and glycosaminoglycans on the cell surface play a key role in the PTD-mediated transduction process.
These PTDs have demonstrated a wide range of transduction behavior. Some studies observed that incubation at 4 °C did not abrogate PTD-mediated transduction nor change the intracellular distribution of the PTD-conjugated proteins (9–11). Other studies, however, reported controversial data that transduction at 4 °C was significantly reduced or even inhibited in comparison with that at 37 °C for TAT, ANTP, and VP22 peptides (12–18). Preincubation of cells with heparinase-III specifically digesting heparan sulfate chains decreased PTD-mediated protein uptake, revealing the role of heparan sulfate on the cell surface (19). Use of mutant cells defective in glycosaminoglycan synthesis provided further evidence to support the hypothesis of PTD binding with proteoglycans (16,20). However, inconsistent data were reported that although acidic proteoglycans formed a pool of charge for PTD binding, this binding had no correlation with the PTD-mediated protein transduction (21).
Experiments were also designed to differentiate these PTD-mediated protein transduction pathways. Mutant cells defective in expressing surface receptors and proteins (LRP, clathrin, caveolin, dynamin, and others) as well as inhibitors selectively blocking the protein uptake routes have been examined, and clathrin-dependent endocytosis (17,22), caveolae-dependent endocytosis (12), macropinocytosis (13,23), and direct penetration mechanism (10) have been concluded. It was also proposed that different cell lines might use different endocytic pathways (24). Simultaneous involvement of different endocytic pathways in one transduction event was also speculated (25). Furthermore, therapeutic applications of these PTD peptides have been examined because they demonstrated an enhanced delivery of functional proteins into cells in vivo and in vitro. Typical examples include PRDX6 protein protection against eye lens epithelial cell death and delaying lens opacity (26), BH4 domain of the anti-apoptotic protein Bcl-xl for protection against the radiation-induced cell death (1), tyrosine hydroxylase for curing 6-OHDA-induced Parkinson’s disease (2), α-synuclein for protection against oxidative stress (4), and SOD for healing ischemic brain injury (5).
In previous studies, biotin or fluorescein has been linked to PTD-conjugated proteins to offer a great convenience in monitoring PTD-mediated transduction process (8). These methods, however, raised some concerns regarding the conjugation efficiency, linkage stability, chemical toxicity, fluorescence lifetime, and the integrity of the cargo proteins because chemical linkage might interrupt the cargo protein structures and interfere with biological functions of the cargo proteins. To overcome these drawbacks, we conjugated the TAT peptide directly to the EGFP protein in this study. Enhanced green fluorescent protein (EGFP) emits a strong and stable fluorescence detectable conveniently with a non-invasive approach in living cells and in paraformaldehyde-fixed cells and therefore has been widely used in cellular biology for imaging. In the current study, we used EGFP as a cargo mimic because its molecular size is compatible with the typical proteins of pharmaceutical potential and its highly fluorescent signal offers a good quality of data for measurement as well. We conjugated the TAT peptide to the EGFP protein in different ways and examined the effect of their intrinsic properties on the protein transduction capabilities.
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- Materials and Methods
- Supporting Information
Delivering biopharmaceutical drugs to the targeted cells has been a great challenge because the impermeable plasma membrane presents a natural barrier for these bulky biomolecules to traverse. Protein transduction domain (PTD) or cell-penetrating peptide (CPP) sheds light on the enhancement of biopharmaceutical molecule delivery (6–8). Many previous studies have examined the interactions of the PTD peptides with surface receptors and proteoglycans on cell membrane and have differentiated the PTD-mediated transduction pathways using different mutant cell lines as well as different inhibitors. To improve the utility of the PTD-protein conjugates for practical applications, we focused our attention to the intrinsic properties of the PTD peptide to examine the effect of the amino acid sequence, the secondary structure, and the relative positions of the TAT peptide with respect to the cargo protein on the TAT-mediated transduction efficiency.
Figure 3 shows that the CD spectra of EGFP and four TAT–EGFP conjugates in the region of 190–240 nm are almost identical. A broad wave trough indicates that β-sheet is the major component of their secondary structures, which is consistent with the crystallographic data of EGFP (28). In addition, we have used bioinformatic analysis to predict that four TAT–EGFP conjugates should have a similar secondary structure (Table S1). These experimental and calculated data suggested that conjugating the TAT peptide to cargo proteins did not alter the native structure of the cargo proteins, and consequently, it could be expected that the TAT-conjugated cargo proteins would demonstrate same biological functions. Flow cytometric results (Figures 4 and 5) showed clearly that the TAT–EGFP conjugates could be delivered into HeLa cells effectively whereas EGFP could not. This comparison proved the utility of the TAT peptide as an effective vehicle to deliver bulky proteins into cells. Furthermore, the TAT-mediated transduction showed a concentration-dependent behavior (Figure 4), being consistent with other observations (4,29). Figure 5 shows that four TAT–EGFP conjugates have been delivered into cells with almost equal efficiency at 37 °C after 60 min of incubation, which leads to an important conclusion that the TAT-mediated transduction does not depend on the sequence orientation of the TAT peptide as well as its relative position. Our results reached a good consistence with previous reports. Ryu et al. have constructed TAT–GFP fusion proteins in which the TAT sequence was fused with the N- and/or C-termini of GFP and observed that the GFP fusion protein with TAT sequence at its C-terminus was taken up as efficiently as the GFP fusion protein with TAT sequence at its N-terminus. When the protein was conjugated to PTDs at its both termini, delivering the fusion proteins was even more efficient (18).
In the current study, incubation of these TAT–EGFP conjugates with HeLa and PC12 cells for 5 days did not show any sign of cell death, offering an experimental evidence of therapeutic feasibility. Previously, Tsutsumi et al. have evaluated the cytotoxicity of four major PTDs (Tat, Antp, Rev, and VP22) in various cell lines (HeLa, HaCaT, A431, Jurkat, MOLT-4, and HL60 cells) and found that the TAT-conjugated protein was the least toxic among the four PTDs (13). Although the reason for this phenomenon is not clear, it has been speculated that the primary structure of the individual PTDs or the cell surface proteins that interact with the individual PTDs might be responsible for the differences in their transduction efficiency and cytotoxicity (13). Recently, Holm et al. have constructed retro-inversion of the two most commonly used CPPs (RI-CPPs) using D-amino acids and examined their cytotoxicity in comparison with their parent peptides in different cell lines (30). Interestingly, treatment of cells with these RI-CPPs induced trypsin insensitivity and rapid severe toxicity in contrast to l-peptides. The reduced metabolic activity, condensed cell nuclei, and the induced apoptosis were evidenced at 20 μm concentration of RI-CPPs within 4 h while parent l-peptides had negligible effects.
To prove the utility of PTD peptides in pharmaceutical applications, an in vivo experiment was conducted where EGFP and the TAT(+)–EGFP conjugate solution were injected intraperitoneally into live mice, respectively. Only the TAT(+)–EGFP conjugate was tested in this experiment because four TAT–EGFP conjugates have already shown an almost equal transduction efficiency in vitro. The intraperitoneal injection of PTD-conjugated functional proteins has shown to be an effective delivery system to the different organs and successful treatment of protection against oxidative stress (4) and against ischemic brain injury (5). Slides of liver, heart, brain, and kidney of these mice were obtained and their fluorescent images were compared. The total fluorescent intensity of images A2–D2 in Figure 7 exhibited at least tenfold increases in comparison with A3–D3 in Figure 7, proving a successful delivery of the TAT–EGFP conjugates to these organs. Fluorescent images A2–D2 were quantified to be 22 000, 24 000, 35 000, and 51 000, respectively. These variations suggested that the TAT-mediated delivery efficiency might be tissue dependent. In a previous study, the TAT peptide was linked to different proteins (β-galactosidase, horseradish peroxidase, and RNase A) and injected intravenously via the tail vein of mice. Staining showed that TAT-β-galactosidase had a high delivery efficiency to heart, liver, and spleen, a low-to-moderate delivery efficiency to lung and skeletal muscle, and little or no delivery to kidney and brain (31). Another study showed that after s.c. injection into mice, a 12-mer peptide (HN-1) was able to cross plasma membranes in a cell-specific manner, recognizing only human head and neck squamous cell cancer (HNSCC) (32).
Our study furnished a valuable and important opinion for the biopharmaceutical agent development and therapeutic applications. Previous studies have showed that PTD peptides were fused with the cargo proteins at its N-terminus and that the PTD sequences were adapted directly from their original sequence in most PTD–protein conjugates. Such conjugates, however, need to be optimized to accomplish desired biological functions. For example, to exert the expected therapeutic purpose in the targeted cells, a protein/peptide-based biopharmaceutical agent needs to recognize the targeted cells correctly, to traverse cellular membrane effectively, and to interact with targeted molecules (DNAs, RNAs, or proteins) in cytoplasm domain or nuclei or mitochondria. Thus, these biopharmaceutical agents should conjugate a cell-recognition sequence, a PTD sequence, and functional proteins or peptides in one construct in a more well-designed manner. Our current data proved that the TAT peptide could be conjugated with functional peptides or proteins in a more flexible way, either in original or in reversed sequence, or in either the N-terminus or the C-terminus of the functional peptide or protein, without scarifying the transduction efficiencies.
The mechanism of PTD-mediated cellular entry has been proposed to be the ionic interaction between the PTDs and the plasma membrane constituents. Most PTDs are rich in basic amino acids, and their isoelectric points are close to each other with a small margin. In the current study, bioinformatic analysis showed that the isoelectric points of TAT–EGFP conjugates were elevated from 5.505 of EGFP protein to 6.885, although the extra eight basic residues of the TAT sequence (YGRKKRRQRRR) accounted for only 5.3% of the total amino acid residues of the TAT–EGFP conjugates. The increased pI values could be used to elucidate the protein transduction mediated by this basic peptide. The high binding affinity between PTD peptides and sulfated glycans on cell membrane has been considered as a driving force to initiate this transduction, and deletion or substitution of a single basic amino acid residue could reduce the transduction efficiency of the PTD peptides (21). Other studies have shown transduction efficiency variations from PTD to PTD, which have been attributed to the differences in the charge distribution, the amphipathicity, the unfolding degree, the polarity, and the molecular shape of the peptides (4,13).
In summary, four TAT-conjugated proteins of TAT(+)–EGFP, TAT(−)–EGFP, EGFP–TAT(+), and EGFP–TAT(−) demonstrate almost identical transduction efficiencies to traverse the cell membrane in vitro, and a capability of delivering into different organs of living mice in vivo. Experimental results and bioinformatic analyses suggest that the TAT-mediated protein transduction is independent of the secondary structure, the amino acid sequence, and the relative location of the TAT peptide. These data conclude that PTD peptide and cargo proteins can be conjugated in more convenient arrangements to meet the requirement for biopharmaceutical applications.