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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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Macropinocytotic inhibitors and TAT PTD-Cre transduction in glycan-deficient cells. A, CHO-K1, pgsA, and Lec2 cells with a stably integrated LSL-GFP construct treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 h. Cells were then treated with TAT PTD-Cre protein in the presence of amiloride for 1 h, followed by trypsinization and replating. GFP expression was assayed by flow cytometry at 24 h after TAT PTD-Cre addition. Inset, cell viability as assayed by flow cytometry immediately following TAT PTD-Cre treatment. B, treatment with wortmannin, a kinase inhibitor, as described for A. C, treatment with cytochalasin D, an inhibitor of F-actin and macropinocytosis, as described for A. D, depletion of cell-surface proteins by trypsin. CHO-K1 LSL cells were treated with trypsin or cell dissociation solution (CDS) for 30 min, followed by washes with PBS and trypsin inhibitor and treatment with TAT PTD-Cre for 1 h. Cells were then washed, trypsinized, and replated. GFP expression was assayed by FACS at 24 h following TAT PTD-Cre treatment. WT, wild-type.
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Figure 4: Macropinocytotic inhibitors and TAT PTD-Cre transduction in glycan-deficient cells. A, CHO-K1, pgsA, and Lec2 cells with a stably integrated LSL-GFP construct treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 h. Cells were then treated with TAT PTD-Cre protein in the presence of amiloride for 1 h, followed by trypsinization and replating. GFP expression was assayed by flow cytometry at 24 h after TAT PTD-Cre addition. Inset, cell viability as assayed by flow cytometry immediately following TAT PTD-Cre treatment. B, treatment with wortmannin, a kinase inhibitor, as described for A. C, treatment with cytochalasin D, an inhibitor of F-actin and macropinocytosis, as described for A. D, depletion of cell-surface proteins by trypsin. CHO-K1 LSL cells were treated with trypsin or cell dissociation solution (CDS) for 30 min, followed by washes with PBS and trypsin inhibitor and treatment with TAT PTD-Cre for 1 h. Cells were then washed, trypsinized, and replated. GFP expression was assayed by FACS at 24 h following TAT PTD-Cre treatment. WT, wild-type.

Mentions: We measured the effect of several macropinocytotic chemical inhibitors on TAT PTD-Cre transduction. Wild-type, HS-deficient, and SA-deficient CHO cells were treated with three different macropinocytotic inhibitors: amiloride (a proton pump inhibitor that specifically affects macropinocytosis) (Fig. 4A), wortmannin (a kinase inhibitor) (Fig. 4B), and cytochalasin D (an inhibitor of F-actin polymerization) (Fig. 4C). Treatment with all three macropinocytotic inhibitors resulted in dramatic reductions of TAT PTD-Cre transduction into cells as measured by a dose-dependent decrease in TAT PTD-Cre-mediated recombination of the LSL-GFP reporter in the absence of significant cellular toxicity (Fig. 4, A–C, insets). In addition, enzymatic removal of cell-surface proteins by trypsin also potently inhibited TAT PTD-Cre-mediated transduction into cells (Fig. 4D). In contrast, nonenzymatic release of cells from tissue culture dishes by cell dissociation solution did not alter TAT-Cre uptake.


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

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

Macropinocytotic inhibitors and TAT PTD-Cre transduction in glycan-deficient cells. A, CHO-K1, pgsA, and Lec2 cells with a stably integrated LSL-GFP construct treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 h. Cells were then treated with TAT PTD-Cre protein in the presence of amiloride for 1 h, followed by trypsinization and replating. GFP expression was assayed by flow cytometry at 24 h after TAT PTD-Cre addition. Inset, cell viability as assayed by flow cytometry immediately following TAT PTD-Cre treatment. B, treatment with wortmannin, a kinase inhibitor, as described for A. C, treatment with cytochalasin D, an inhibitor of F-actin and macropinocytosis, as described for A. D, depletion of cell-surface proteins by trypsin. CHO-K1 LSL cells were treated with trypsin or cell dissociation solution (CDS) for 30 min, followed by washes with PBS and trypsin inhibitor and treatment with TAT PTD-Cre for 1 h. Cells were then washed, trypsinized, and replated. GFP expression was assayed by FACS at 24 h following TAT PTD-Cre treatment. WT, wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Macropinocytotic inhibitors and TAT PTD-Cre transduction in glycan-deficient cells. A, CHO-K1, pgsA, and Lec2 cells with a stably integrated LSL-GFP construct treated with the indicated concentrations of amiloride, an inhibitor of macropinocytosis, for 1 h. Cells were then treated with TAT PTD-Cre protein in the presence of amiloride for 1 h, followed by trypsinization and replating. GFP expression was assayed by flow cytometry at 24 h after TAT PTD-Cre addition. Inset, cell viability as assayed by flow cytometry immediately following TAT PTD-Cre treatment. B, treatment with wortmannin, a kinase inhibitor, as described for A. C, treatment with cytochalasin D, an inhibitor of F-actin and macropinocytosis, as described for A. D, depletion of cell-surface proteins by trypsin. CHO-K1 LSL cells were treated with trypsin or cell dissociation solution (CDS) for 30 min, followed by washes with PBS and trypsin inhibitor and treatment with TAT PTD-Cre for 1 h. Cells were then washed, trypsinized, and replated. GFP expression was assayed by FACS at 24 h following TAT PTD-Cre treatment. WT, wild-type.
Mentions: We measured the effect of several macropinocytotic chemical inhibitors on TAT PTD-Cre transduction. Wild-type, HS-deficient, and SA-deficient CHO cells were treated with three different macropinocytotic inhibitors: amiloride (a proton pump inhibitor that specifically affects macropinocytosis) (Fig. 4A), wortmannin (a kinase inhibitor) (Fig. 4B), and cytochalasin D (an inhibitor of F-actin polymerization) (Fig. 4C). Treatment with all three macropinocytotic inhibitors resulted in dramatic reductions of TAT PTD-Cre transduction into cells as measured by a dose-dependent decrease in TAT PTD-Cre-mediated recombination of the LSL-GFP reporter in the absence of significant cellular toxicity (Fig. 4, A–C, insets). In addition, enzymatic removal of cell-surface proteins by trypsin also potently inhibited TAT PTD-Cre-mediated transduction into cells (Fig. 4D). In contrast, nonenzymatic release of cells from tissue culture dishes by cell dissociation solution did not alter TAT-Cre uptake.

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