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Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.

Kwon SJ, Han K, Jung S, Lee JE, Park S, Cheon YP, Lim HJ - BMC Biotechnol. (2009)

Bottom Line: The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction.Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP.MPG-EGFP transduction is also observed in the mammalian oocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Science & Technology IBST Konkuk University 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Korea. ksjpo47@hanmail.net

ABSTRACT

Background: MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction.

Results: We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes.

Conclusion: The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.

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Efficient transduction of the MPG-EGFP fusion protein into various cell lines. A. 40 μg/ml MPG-EGFP was added to AN3CA or HeLa cells. NIH3T3, BV2, 293T, and HT29 were also tested (data not shown). MPG-EGFP exhibits a punctate vesicular pattern in the cytoplasm in cells. Fixation of the cells with 4% PFA (fixed) did not alter the subcellular distribution of MPG-EGFP. Cells were all observed under a confocal microscope. Fixed cells were counterstained with TO-PRO-3 iodide (shown in blue, 1:500). B. MPG-EGFP is mostly present in the endosomes. Cells were stained with 5 μg/ml FM4-64, a general marker of endocytosis (shown in red), and visualized under a confocal microscope without fixation. Overlapping of MPG-EGFP signal and FM4-64 staining generates a yellow fluorescence. C. Vesicle fractionation was performed using HeLa cells treated with EGFP or MPG-EGFP for 1 hr. -, no treatment; EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. Western blotting was performed with anti-GFP antibody.
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Figure 1: Efficient transduction of the MPG-EGFP fusion protein into various cell lines. A. 40 μg/ml MPG-EGFP was added to AN3CA or HeLa cells. NIH3T3, BV2, 293T, and HT29 were also tested (data not shown). MPG-EGFP exhibits a punctate vesicular pattern in the cytoplasm in cells. Fixation of the cells with 4% PFA (fixed) did not alter the subcellular distribution of MPG-EGFP. Cells were all observed under a confocal microscope. Fixed cells were counterstained with TO-PRO-3 iodide (shown in blue, 1:500). B. MPG-EGFP is mostly present in the endosomes. Cells were stained with 5 μg/ml FM4-64, a general marker of endocytosis (shown in red), and visualized under a confocal microscope without fixation. Overlapping of MPG-EGFP signal and FM4-64 staining generates a yellow fluorescence. C. Vesicle fractionation was performed using HeLa cells treated with EGFP or MPG-EGFP for 1 hr. -, no treatment; EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. Western blotting was performed with anti-GFP antibody.

Mentions: We initially chose 8 CPPs that have not been used to deliver protein cargoes in the form of fusion proteins (Table 1), and prepared CPP-EGFP fusion proteins. These CPPs, in peptide forms, had all been shown effective in delivering oligonucleotides or nucleic acids into cells when used as a mixture. As N-terminal tags of EGFP recombinant proteins, however, most of these CPPs did not seem to enter cells efficiently (data not shown). Among the tested CPPs, MPG-EGFP entered the cells and exhibited a punctate vesicular pattern of EGFP fluorescence (Figure 1A). Efficient transduction of MPG-EGFP was confirmed in various cell lines, including AN3CA, 293T, NIH3T3, F9, BV2, and HT29. Treatment of MPG-EGFP at 40, 80, or 120 μg/ml for more than 24 hr did not cause any significant cytotoxicity (all above 93% survival rate).


Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.

Kwon SJ, Han K, Jung S, Lee JE, Park S, Cheon YP, Lim HJ - BMC Biotechnol. (2009)

Efficient transduction of the MPG-EGFP fusion protein into various cell lines. A. 40 μg/ml MPG-EGFP was added to AN3CA or HeLa cells. NIH3T3, BV2, 293T, and HT29 were also tested (data not shown). MPG-EGFP exhibits a punctate vesicular pattern in the cytoplasm in cells. Fixation of the cells with 4% PFA (fixed) did not alter the subcellular distribution of MPG-EGFP. Cells were all observed under a confocal microscope. Fixed cells were counterstained with TO-PRO-3 iodide (shown in blue, 1:500). B. MPG-EGFP is mostly present in the endosomes. Cells were stained with 5 μg/ml FM4-64, a general marker of endocytosis (shown in red), and visualized under a confocal microscope without fixation. Overlapping of MPG-EGFP signal and FM4-64 staining generates a yellow fluorescence. C. Vesicle fractionation was performed using HeLa cells treated with EGFP or MPG-EGFP for 1 hr. -, no treatment; EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. Western blotting was performed with anti-GFP antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748071&req=5

Figure 1: Efficient transduction of the MPG-EGFP fusion protein into various cell lines. A. 40 μg/ml MPG-EGFP was added to AN3CA or HeLa cells. NIH3T3, BV2, 293T, and HT29 were also tested (data not shown). MPG-EGFP exhibits a punctate vesicular pattern in the cytoplasm in cells. Fixation of the cells with 4% PFA (fixed) did not alter the subcellular distribution of MPG-EGFP. Cells were all observed under a confocal microscope. Fixed cells were counterstained with TO-PRO-3 iodide (shown in blue, 1:500). B. MPG-EGFP is mostly present in the endosomes. Cells were stained with 5 μg/ml FM4-64, a general marker of endocytosis (shown in red), and visualized under a confocal microscope without fixation. Overlapping of MPG-EGFP signal and FM4-64 staining generates a yellow fluorescence. C. Vesicle fractionation was performed using HeLa cells treated with EGFP or MPG-EGFP for 1 hr. -, no treatment; EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. Western blotting was performed with anti-GFP antibody.
Mentions: We initially chose 8 CPPs that have not been used to deliver protein cargoes in the form of fusion proteins (Table 1), and prepared CPP-EGFP fusion proteins. These CPPs, in peptide forms, had all been shown effective in delivering oligonucleotides or nucleic acids into cells when used as a mixture. As N-terminal tags of EGFP recombinant proteins, however, most of these CPPs did not seem to enter cells efficiently (data not shown). Among the tested CPPs, MPG-EGFP entered the cells and exhibited a punctate vesicular pattern of EGFP fluorescence (Figure 1A). Efficient transduction of MPG-EGFP was confirmed in various cell lines, including AN3CA, 293T, NIH3T3, F9, BV2, and HT29. Treatment of MPG-EGFP at 40, 80, or 120 μg/ml for more than 24 hr did not cause any significant cytotoxicity (all above 93% survival rate).

Bottom Line: The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction.Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP.MPG-EGFP transduction is also observed in the mammalian oocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Science & Technology IBST Konkuk University 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Korea. ksjpo47@hanmail.net

ABSTRACT

Background: MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction.

Results: We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes.

Conclusion: The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.

Show MeSH
Related in: MedlinePlus