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Revised role of glycosaminoglycans in TAT protein transduction domain-mediated cellular transduction.

Gump JM, June RK, Dowdy SF - J. Biol. Chem. (2009)

Bottom Line: Similar results were obtained in cells where glycans were enzymatically removed.In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction.Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093-0686, USA.

ABSTRACT
Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

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TAT PTD-Cre transduction occurs in the absence of glycosaminoglycans and SAs in parental CHO and glycan-deficient cell lines. A, TAT PTD-Cre recombination in CHO-K1 and derivative glycan mutant pgsA and Lec2 cells with a stably integrated Lox-STOP-Lox-GFP expression construct. Cells were treated with purified recombinant TAT PTD-Cre fusion protein or control recombinant Cre (no TAT PTD) at the indicated concentrations for 1 h, followed by trypsinization and replating. Flow cytometry was performed at 24 h after Cre addition. As indicated, control cells were treated with cytochalasin D to inhibit macropinocytosis for 1 h at 37 °C immediately preceding and during TAT PTD-Cre treatment. B, photomicrographs of TAT PTD-Cre recombination-induced GFP expression in parental and glycan mutant cells with stably integrated LSL-GFP construct. C, FACS profiles of representative samples from the experiment in A. WT, wild-type.
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Figure 3: TAT PTD-Cre transduction occurs in the absence of glycosaminoglycans and SAs in parental CHO and glycan-deficient cell lines. A, TAT PTD-Cre recombination in CHO-K1 and derivative glycan mutant pgsA and Lec2 cells with a stably integrated Lox-STOP-Lox-GFP expression construct. Cells were treated with purified recombinant TAT PTD-Cre fusion protein or control recombinant Cre (no TAT PTD) at the indicated concentrations for 1 h, followed by trypsinization and replating. Flow cytometry was performed at 24 h after Cre addition. As indicated, control cells were treated with cytochalasin D to inhibit macropinocytosis for 1 h at 37 °C immediately preceding and during TAT PTD-Cre treatment. B, photomicrographs of TAT PTD-Cre recombination-induced GFP expression in parental and glycan mutant cells with stably integrated LSL-GFP construct. C, FACS profiles of representative samples from the experiment in A. WT, wild-type.

Mentions: We analyzed TAT PTD-Cre transduction in stable LSL-GFP clones of parental glycan-positive wild-type CHO-K1 and derivative glycan-deficient pgsA and Lec2 cell lines. Treatment of each of these cell lines with TAT PTD-Cre induced GFP expression in a dose-dependent manner as observed by flow cytometry (Fig. 3, A and C) and microscopy (Fig. 3B). This demonstrates the ability of TAT PTD-Cre to enter cells and transit to the nucleus to recombine the LSL genetic element in the absence of HS and SA. Although cells lacking glycans showed a slight shift of the dose-response curve, both HS- and SA-deficient cells reached the same maximal response at 2 μm. Similar results were obtained using recombinant Arg8-PTD-Cre fusion protein (data not shown). In contrast, control recombinant Cre protein (no PTD) showed no induction of GFP above background levels (Fig. 3A). Inhibition of macropinocytosis by cytochalasin D resulted in a dramatic reduction in TAT PTD-Cre-mediated recombination and GFP induction (Fig. 3, A and B).


Revised role of glycosaminoglycans in TAT protein transduction domain-mediated cellular transduction.

Gump JM, June RK, Dowdy SF - J. Biol. Chem. (2009)

TAT PTD-Cre transduction occurs in the absence of glycosaminoglycans and SAs in parental CHO and glycan-deficient cell lines. A, TAT PTD-Cre recombination in CHO-K1 and derivative glycan mutant pgsA and Lec2 cells with a stably integrated Lox-STOP-Lox-GFP expression construct. Cells were treated with purified recombinant TAT PTD-Cre fusion protein or control recombinant Cre (no TAT PTD) at the indicated concentrations for 1 h, followed by trypsinization and replating. Flow cytometry was performed at 24 h after Cre addition. As indicated, control cells were treated with cytochalasin D to inhibit macropinocytosis for 1 h at 37 °C immediately preceding and during TAT PTD-Cre treatment. B, photomicrographs of TAT PTD-Cre recombination-induced GFP expression in parental and glycan mutant cells with stably integrated LSL-GFP construct. C, FACS profiles of representative samples from the experiment in A. WT, wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2801275&req=5

Figure 3: TAT PTD-Cre transduction occurs in the absence of glycosaminoglycans and SAs in parental CHO and glycan-deficient cell lines. A, TAT PTD-Cre recombination in CHO-K1 and derivative glycan mutant pgsA and Lec2 cells with a stably integrated Lox-STOP-Lox-GFP expression construct. Cells were treated with purified recombinant TAT PTD-Cre fusion protein or control recombinant Cre (no TAT PTD) at the indicated concentrations for 1 h, followed by trypsinization and replating. Flow cytometry was performed at 24 h after Cre addition. As indicated, control cells were treated with cytochalasin D to inhibit macropinocytosis for 1 h at 37 °C immediately preceding and during TAT PTD-Cre treatment. B, photomicrographs of TAT PTD-Cre recombination-induced GFP expression in parental and glycan mutant cells with stably integrated LSL-GFP construct. C, FACS profiles of representative samples from the experiment in A. WT, wild-type.
Mentions: We analyzed TAT PTD-Cre transduction in stable LSL-GFP clones of parental glycan-positive wild-type CHO-K1 and derivative glycan-deficient pgsA and Lec2 cell lines. Treatment of each of these cell lines with TAT PTD-Cre induced GFP expression in a dose-dependent manner as observed by flow cytometry (Fig. 3, A and C) and microscopy (Fig. 3B). This demonstrates the ability of TAT PTD-Cre to enter cells and transit to the nucleus to recombine the LSL genetic element in the absence of HS and SA. Although cells lacking glycans showed a slight shift of the dose-response curve, both HS- and SA-deficient cells reached the same maximal response at 2 μm. Similar results were obtained using recombinant Arg8-PTD-Cre fusion protein (data not shown). In contrast, control recombinant Cre protein (no PTD) showed no induction of GFP above background levels (Fig. 3A). Inhibition of macropinocytosis by cytochalasin D resulted in a dramatic reduction in TAT PTD-Cre-mediated recombination and GFP induction (Fig. 3, A and B).

Bottom Line: Similar results were obtained in cells where glycans were enzymatically removed.In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction.Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093-0686, USA.

ABSTRACT
Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.

Show MeSH
Related in: MedlinePlus