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Ganglioside-magnetosome complex formation enhances uptake of gangliosides by cells.

Guan F, Li X, Guo J, Yang G, Li X - Int J Nanomedicine (2015)

Bottom Line: The use of GM1-magnetosome complex resulted in the greatest enhancement of ganglioside incorporation by cells.GM3-magnetosome complex significantly inhibited EGF-induced phosphorylation of the epidermal growth factor receptor.Both of these effects were further enhanced by the presence of a magnetic field.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People's Republic of China.

ABSTRACT
Bacterial magnetosomes, because of their nano-scale size, have a large surface-to-volume ratio and are able to carry large quantities of bioactive substances such as enzymes, antibodies, and genes. Gangliosides, a family of sialic acid-containing glycosphingolipids, function as distinctive cell surface markers and as specific determinants in cellular recognition and cell-to-cell communication. Exogenously added gangliosides are often used to study biological functions, transport mechanisms, and metabolism of their endogenous counterparts. Absorption of gangliosides into cells is typically limited by their tendency to aggregate into micelles in aqueous media. We describe here a simple strategy to remove proteins from the magnetosome membrane by sodium dodecyl sulfate treatment, and efficiently immobilize a ganglioside (GM1 or GM3) on the magnetosome by mild ultrasonic treatment. The maximum of 11.7±1.2 µg GM1 and 11.6±1.5 μg GM3 was loaded onto 1 mg magnetosome, respectively. Complexes of ganglioside-magnetosomes stored at 4°C for certain days presented the consistent stability. The use of GM1-magnetosome complex resulted in the greatest enhancement of ganglioside incorporation by cells. GM3-magnetosome complex significantly inhibited EGF-induced phosphorylation of the epidermal growth factor receptor. Both of these effects were further enhanced by the presence of a magnetic field.

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Related in: MedlinePlus

Effect of GM3 on EGF-induced tyrosine phosphorylation of EGFR.Notes: (A) Technique (schematic) for evaluating interaction of GM3-magnetosomes with EGFR. (B) A431 cell lysate was incubated with PE-magnetosomes or GM3-magnetosomes, separated by a magnetic field, boiled in 50 µL sample buffer, subjected to SDS-PAGE, visualized by silver staining (left), or analyzed by Western blotting (right). Lane 1: A431 cell lysate. Lane 2: lysate incubated with PE-magnetosomes. Lane 3: lysate incubated with GM3-magnetosomes. Lane 4: protein bound to PE-magnetosomes. Lane 5: protein bound to GM3-magnetosomes. (C) Inhibitory effect of GM3 and GM3-magnetosomes on EGFR kinase activity in A431 cells. Amount of GM3 per 1 mg magnetosome =0.104 µg. 10*: concentration of total GM3-magnetosome complex added to medium =10 µg/mL. EGF-induced phosphorylation of EGFR was quantified as density of phospho-EGFR divided by that of EGFR, by densitometry using Image J. Experiments were performed in triplicate, and representative Western blot results are shown (left). Data are shown as mean ± SD (right). *P<0.05; ***P<0.001.Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; p-EGFR, phosphorylated epidermal growth factor receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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f4-ijn-10-6919: Effect of GM3 on EGF-induced tyrosine phosphorylation of EGFR.Notes: (A) Technique (schematic) for evaluating interaction of GM3-magnetosomes with EGFR. (B) A431 cell lysate was incubated with PE-magnetosomes or GM3-magnetosomes, separated by a magnetic field, boiled in 50 µL sample buffer, subjected to SDS-PAGE, visualized by silver staining (left), or analyzed by Western blotting (right). Lane 1: A431 cell lysate. Lane 2: lysate incubated with PE-magnetosomes. Lane 3: lysate incubated with GM3-magnetosomes. Lane 4: protein bound to PE-magnetosomes. Lane 5: protein bound to GM3-magnetosomes. (C) Inhibitory effect of GM3 and GM3-magnetosomes on EGFR kinase activity in A431 cells. Amount of GM3 per 1 mg magnetosome =0.104 µg. 10*: concentration of total GM3-magnetosome complex added to medium =10 µg/mL. EGF-induced phosphorylation of EGFR was quantified as density of phospho-EGFR divided by that of EGFR, by densitometry using Image J. Experiments were performed in triplicate, and representative Western blot results are shown (left). Data are shown as mean ± SD (right). *P<0.05; ***P<0.001.Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; p-EGFR, phosphorylated epidermal growth factor receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mentions: Exogenous addition of gangliosides is a technique often used to elucidate the function of specific gangliosides as second messenger molecules or receptor modulators in cultured cells.7,26,27 For example, exogenous GM3 blocks tyrosine phosphorylation of EGFR in human epidermoid carcinoma A431 and other cell lines.28,29 The absorption of gangliosides into cells is typically limited by their tendency to aggregate into micelles in aqueous media.30 We examined the inhibitory effect of magnetosomes grafted with GM3 (GM3-magnetosome complex) on EGFR activation. A431 cells (high EGFR expressors) are often used for studying mechanisms whereby the EGFR signal pathway is turned on or off.28 We confirmed the interaction of GM3 with EGFR using GM3-magnetosome complex. A431 cell lysate was incubated with the complex, and specific proteins binding to the complex were separated by magnetic field (Figure 4A). The EGFR protein in A431 cell lysate was clearly pulled down by GM3-magnetosome complex and confirmed by Western blotting, whereas phosphatidylethanolamine-magnetosomes complex had no such effect (Figure 4B). EGF stimulation activated pEGFR expression as expected, and such activation was inhibited by GM3. The inhibitory effect of GM3 on pEGFR activation was significantly enhanced by complexing with magnetosomes, especially together with the presence of a magnetic field (Figure 4C).


Ganglioside-magnetosome complex formation enhances uptake of gangliosides by cells.

Guan F, Li X, Guo J, Yang G, Li X - Int J Nanomedicine (2015)

Effect of GM3 on EGF-induced tyrosine phosphorylation of EGFR.Notes: (A) Technique (schematic) for evaluating interaction of GM3-magnetosomes with EGFR. (B) A431 cell lysate was incubated with PE-magnetosomes or GM3-magnetosomes, separated by a magnetic field, boiled in 50 µL sample buffer, subjected to SDS-PAGE, visualized by silver staining (left), or analyzed by Western blotting (right). Lane 1: A431 cell lysate. Lane 2: lysate incubated with PE-magnetosomes. Lane 3: lysate incubated with GM3-magnetosomes. Lane 4: protein bound to PE-magnetosomes. Lane 5: protein bound to GM3-magnetosomes. (C) Inhibitory effect of GM3 and GM3-magnetosomes on EGFR kinase activity in A431 cells. Amount of GM3 per 1 mg magnetosome =0.104 µg. 10*: concentration of total GM3-magnetosome complex added to medium =10 µg/mL. EGF-induced phosphorylation of EGFR was quantified as density of phospho-EGFR divided by that of EGFR, by densitometry using Image J. Experiments were performed in triplicate, and representative Western blot results are shown (left). Data are shown as mean ± SD (right). *P<0.05; ***P<0.001.Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; p-EGFR, phosphorylated epidermal growth factor receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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f4-ijn-10-6919: Effect of GM3 on EGF-induced tyrosine phosphorylation of EGFR.Notes: (A) Technique (schematic) for evaluating interaction of GM3-magnetosomes with EGFR. (B) A431 cell lysate was incubated with PE-magnetosomes or GM3-magnetosomes, separated by a magnetic field, boiled in 50 µL sample buffer, subjected to SDS-PAGE, visualized by silver staining (left), or analyzed by Western blotting (right). Lane 1: A431 cell lysate. Lane 2: lysate incubated with PE-magnetosomes. Lane 3: lysate incubated with GM3-magnetosomes. Lane 4: protein bound to PE-magnetosomes. Lane 5: protein bound to GM3-magnetosomes. (C) Inhibitory effect of GM3 and GM3-magnetosomes on EGFR kinase activity in A431 cells. Amount of GM3 per 1 mg magnetosome =0.104 µg. 10*: concentration of total GM3-magnetosome complex added to medium =10 µg/mL. EGF-induced phosphorylation of EGFR was quantified as density of phospho-EGFR divided by that of EGFR, by densitometry using Image J. Experiments were performed in triplicate, and representative Western blot results are shown (left). Data are shown as mean ± SD (right). *P<0.05; ***P<0.001.Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; p-EGFR, phosphorylated epidermal growth factor receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Mentions: Exogenous addition of gangliosides is a technique often used to elucidate the function of specific gangliosides as second messenger molecules or receptor modulators in cultured cells.7,26,27 For example, exogenous GM3 blocks tyrosine phosphorylation of EGFR in human epidermoid carcinoma A431 and other cell lines.28,29 The absorption of gangliosides into cells is typically limited by their tendency to aggregate into micelles in aqueous media.30 We examined the inhibitory effect of magnetosomes grafted with GM3 (GM3-magnetosome complex) on EGFR activation. A431 cells (high EGFR expressors) are often used for studying mechanisms whereby the EGFR signal pathway is turned on or off.28 We confirmed the interaction of GM3 with EGFR using GM3-magnetosome complex. A431 cell lysate was incubated with the complex, and specific proteins binding to the complex were separated by magnetic field (Figure 4A). The EGFR protein in A431 cell lysate was clearly pulled down by GM3-magnetosome complex and confirmed by Western blotting, whereas phosphatidylethanolamine-magnetosomes complex had no such effect (Figure 4B). EGF stimulation activated pEGFR expression as expected, and such activation was inhibited by GM3. The inhibitory effect of GM3 on pEGFR activation was significantly enhanced by complexing with magnetosomes, especially together with the presence of a magnetic field (Figure 4C).

Bottom Line: The use of GM1-magnetosome complex resulted in the greatest enhancement of ganglioside incorporation by cells.GM3-magnetosome complex significantly inhibited EGF-induced phosphorylation of the epidermal growth factor receptor.Both of these effects were further enhanced by the presence of a magnetic field.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People's Republic of China.

ABSTRACT
Bacterial magnetosomes, because of their nano-scale size, have a large surface-to-volume ratio and are able to carry large quantities of bioactive substances such as enzymes, antibodies, and genes. Gangliosides, a family of sialic acid-containing glycosphingolipids, function as distinctive cell surface markers and as specific determinants in cellular recognition and cell-to-cell communication. Exogenously added gangliosides are often used to study biological functions, transport mechanisms, and metabolism of their endogenous counterparts. Absorption of gangliosides into cells is typically limited by their tendency to aggregate into micelles in aqueous media. We describe here a simple strategy to remove proteins from the magnetosome membrane by sodium dodecyl sulfate treatment, and efficiently immobilize a ganglioside (GM1 or GM3) on the magnetosome by mild ultrasonic treatment. The maximum of 11.7±1.2 µg GM1 and 11.6±1.5 μg GM3 was loaded onto 1 mg magnetosome, respectively. Complexes of ganglioside-magnetosomes stored at 4°C for certain days presented the consistent stability. The use of GM1-magnetosome complex resulted in the greatest enhancement of ganglioside incorporation by cells. GM3-magnetosome complex significantly inhibited EGF-induced phosphorylation of the epidermal growth factor receptor. Both of these effects were further enhanced by the presence of a magnetic field.

No MeSH data available.


Related in: MedlinePlus