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Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

Vaidyanath A, Hashizume T, Nagaoka T, Takeyasu N, Satoh H, Chen L, Wang J, Kasai T, Kudoh T, Satoh A, Fu L, Seno M - J. Cell. Mol. Med. (2011)

Bottom Line: Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand.Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells.Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nano-Biotechnology, Department of Medical Bioengineering Science, Graduate School of Natural Science and Biotechnology, Okayama University, Kita-ku, Okayama, Japan.

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Characterization of BNC displaying EC-Fc. (A) Schematic representation of the multivalent display of EC-Fc on ZZ-BNC (EC-Fc/BNC) and Fc on ZZ-BNC (Fc/BNC). (B) The solubility of BNC-displaying Fc/BNC and IgG/BNC was evaluated with FITC-labelled BNC. Residual fluorescence in supernatant was measured in varying molecular ratio of Fc or human IgG to ZZ-BNC. The intensity from the FITC-labelled ZZ-BNC without Fc was calculated as 1 in each graph. (C) Western blot analysis of Fc in the supernatant obtained in (B). Fc/BNC in the supernatant was immunoprecipitated with anti-HBsAg antibody conjugated to micro beads and was subjected to Western blotting. The Fc on the blot was detected with anti-human IgG. The bands were densitometrically analysed with ImageJ and relative intensity of each lane was plotted. (D–F) Assessment of internalization of EC-Fc/BNC in SK-BR-3 cells through Western blot. (D) EC-Fc/BNC (40 nM/2 nM) or Fc/BNC (40 nM/2 nM) was incubated with SK-BR-3 cells for 5 hrs at 4°C and 37°C. (E) SK-BR-3 cells were treated with various concentration of EC-Fc/BNC from 1 to 10 nM. Fc/BNC in 10 nM was taken as control. (F) SK-BR-3 cells were treated with 2 nM EC-Fc/BNC at various time periods. Simultaneously 2 nM Fc/BNC was taken as control. (D–E) After the incubation the cells were trypsinised and lysed followed by immunoprecipitation with anti-HBsAg antibody conjugated to micro beads. The precipitates were immunoblotted and were detected with anti–pre-S1 antibody. The bands of BNC were densitometrically analysed by ImageJ and plotted into each graph to evaluate amount endocytosed. (G, H) Confocal microscopic observation of SK-BR-3 cells treated with EC-Fc/BNC or Fc/BNC. Cells were incubated for various time periods (G) and for 4 hrs (H). The RITC-labelled ZZ-BNC was used and the cells were fixed and permeabilized. EC-Fc or Fc were detected with anti-human IgG labelled with FITC (G) and ErbB2 was detected with sc-08 antibody followed by rabbit anti-mouse IgG Alexa 488 (H). Bars, 10 μm.
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fig03: Characterization of BNC displaying EC-Fc. (A) Schematic representation of the multivalent display of EC-Fc on ZZ-BNC (EC-Fc/BNC) and Fc on ZZ-BNC (Fc/BNC). (B) The solubility of BNC-displaying Fc/BNC and IgG/BNC was evaluated with FITC-labelled BNC. Residual fluorescence in supernatant was measured in varying molecular ratio of Fc or human IgG to ZZ-BNC. The intensity from the FITC-labelled ZZ-BNC without Fc was calculated as 1 in each graph. (C) Western blot analysis of Fc in the supernatant obtained in (B). Fc/BNC in the supernatant was immunoprecipitated with anti-HBsAg antibody conjugated to micro beads and was subjected to Western blotting. The Fc on the blot was detected with anti-human IgG. The bands were densitometrically analysed with ImageJ and relative intensity of each lane was plotted. (D–F) Assessment of internalization of EC-Fc/BNC in SK-BR-3 cells through Western blot. (D) EC-Fc/BNC (40 nM/2 nM) or Fc/BNC (40 nM/2 nM) was incubated with SK-BR-3 cells for 5 hrs at 4°C and 37°C. (E) SK-BR-3 cells were treated with various concentration of EC-Fc/BNC from 1 to 10 nM. Fc/BNC in 10 nM was taken as control. (F) SK-BR-3 cells were treated with 2 nM EC-Fc/BNC at various time periods. Simultaneously 2 nM Fc/BNC was taken as control. (D–E) After the incubation the cells were trypsinised and lysed followed by immunoprecipitation with anti-HBsAg antibody conjugated to micro beads. The precipitates were immunoblotted and were detected with anti–pre-S1 antibody. The bands of BNC were densitometrically analysed by ImageJ and plotted into each graph to evaluate amount endocytosed. (G, H) Confocal microscopic observation of SK-BR-3 cells treated with EC-Fc/BNC or Fc/BNC. Cells were incubated for various time periods (G) and for 4 hrs (H). The RITC-labelled ZZ-BNC was used and the cells were fixed and permeabilized. EC-Fc or Fc were detected with anti-human IgG labelled with FITC (G) and ErbB2 was detected with sc-08 antibody followed by rabbit anti-mouse IgG Alexa 488 (H). Bars, 10 μm.

Mentions: The multivalent form of EC-1 peptide was prepared exploiting the affinity of ZZ-BNC for IgG Fc region (Fig. 3A). When ZZ-BNC was mixed with EC-Fc, the ligand EC-1 was multivalently displayed on the surface of ZZ-BNC (EC-Fc/BNC). On the other hand, ZZ-BNC displaying ligand was just prepared by mixing Fc protein with ZZ- BNC (Fc/BNC). First of all, binding capacity and binding efficiency of the ZZ-BNC with ligand was optimized and characterized. To optimize the ratio of Fc fusion molecule to BNC, FITC-labelled ZZ-BNC was mixed with Fc protein at variable molar ratios of 1:10, 1:20, 1:40, 1:60, 1:80 and 1:100, respectively, and the residual fluorescent intensity in the supernatant was measured. As shown in Figure 3B, the soluble Fc/BNC was reduced to 30% at a molar ratio of ZZ-BNC to Fc protein at 1:100 when judged from the fluorescence in the supernatant. Similarly, when human IgG was used, the soluble IgG/BNC was found reduced to 10% at a molar ratio of ZZ-BNC to IgG protein at 1:100. The amount of Fc protein bound to ZZ-BNC in the supernatant was further estimated from intensity of the band detected by Western blotting (Fig. 3C). As the result, the amount of Fc protein bound to ZZ-BNC was determined maximum when the molar ratio of ZZ-BNC to Fc protein was at 1:20. Thus, EC-Fc/ZZ-BNC was prepared by mixing ZZ-BNC and EC-Fc at the molar ratio of 1:20 for further experiments.


Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.

Vaidyanath A, Hashizume T, Nagaoka T, Takeyasu N, Satoh H, Chen L, Wang J, Kasai T, Kudoh T, Satoh A, Fu L, Seno M - J. Cell. Mol. Med. (2011)

Characterization of BNC displaying EC-Fc. (A) Schematic representation of the multivalent display of EC-Fc on ZZ-BNC (EC-Fc/BNC) and Fc on ZZ-BNC (Fc/BNC). (B) The solubility of BNC-displaying Fc/BNC and IgG/BNC was evaluated with FITC-labelled BNC. Residual fluorescence in supernatant was measured in varying molecular ratio of Fc or human IgG to ZZ-BNC. The intensity from the FITC-labelled ZZ-BNC without Fc was calculated as 1 in each graph. (C) Western blot analysis of Fc in the supernatant obtained in (B). Fc/BNC in the supernatant was immunoprecipitated with anti-HBsAg antibody conjugated to micro beads and was subjected to Western blotting. The Fc on the blot was detected with anti-human IgG. The bands were densitometrically analysed with ImageJ and relative intensity of each lane was plotted. (D–F) Assessment of internalization of EC-Fc/BNC in SK-BR-3 cells through Western blot. (D) EC-Fc/BNC (40 nM/2 nM) or Fc/BNC (40 nM/2 nM) was incubated with SK-BR-3 cells for 5 hrs at 4°C and 37°C. (E) SK-BR-3 cells were treated with various concentration of EC-Fc/BNC from 1 to 10 nM. Fc/BNC in 10 nM was taken as control. (F) SK-BR-3 cells were treated with 2 nM EC-Fc/BNC at various time periods. Simultaneously 2 nM Fc/BNC was taken as control. (D–E) After the incubation the cells were trypsinised and lysed followed by immunoprecipitation with anti-HBsAg antibody conjugated to micro beads. The precipitates were immunoblotted and were detected with anti–pre-S1 antibody. The bands of BNC were densitometrically analysed by ImageJ and plotted into each graph to evaluate amount endocytosed. (G, H) Confocal microscopic observation of SK-BR-3 cells treated with EC-Fc/BNC or Fc/BNC. Cells were incubated for various time periods (G) and for 4 hrs (H). The RITC-labelled ZZ-BNC was used and the cells were fixed and permeabilized. EC-Fc or Fc were detected with anti-human IgG labelled with FITC (G) and ErbB2 was detected with sc-08 antibody followed by rabbit anti-mouse IgG Alexa 488 (H). Bars, 10 μm.
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fig03: Characterization of BNC displaying EC-Fc. (A) Schematic representation of the multivalent display of EC-Fc on ZZ-BNC (EC-Fc/BNC) and Fc on ZZ-BNC (Fc/BNC). (B) The solubility of BNC-displaying Fc/BNC and IgG/BNC was evaluated with FITC-labelled BNC. Residual fluorescence in supernatant was measured in varying molecular ratio of Fc or human IgG to ZZ-BNC. The intensity from the FITC-labelled ZZ-BNC without Fc was calculated as 1 in each graph. (C) Western blot analysis of Fc in the supernatant obtained in (B). Fc/BNC in the supernatant was immunoprecipitated with anti-HBsAg antibody conjugated to micro beads and was subjected to Western blotting. The Fc on the blot was detected with anti-human IgG. The bands were densitometrically analysed with ImageJ and relative intensity of each lane was plotted. (D–F) Assessment of internalization of EC-Fc/BNC in SK-BR-3 cells through Western blot. (D) EC-Fc/BNC (40 nM/2 nM) or Fc/BNC (40 nM/2 nM) was incubated with SK-BR-3 cells for 5 hrs at 4°C and 37°C. (E) SK-BR-3 cells were treated with various concentration of EC-Fc/BNC from 1 to 10 nM. Fc/BNC in 10 nM was taken as control. (F) SK-BR-3 cells were treated with 2 nM EC-Fc/BNC at various time periods. Simultaneously 2 nM Fc/BNC was taken as control. (D–E) After the incubation the cells were trypsinised and lysed followed by immunoprecipitation with anti-HBsAg antibody conjugated to micro beads. The precipitates were immunoblotted and were detected with anti–pre-S1 antibody. The bands of BNC were densitometrically analysed by ImageJ and plotted into each graph to evaluate amount endocytosed. (G, H) Confocal microscopic observation of SK-BR-3 cells treated with EC-Fc/BNC or Fc/BNC. Cells were incubated for various time periods (G) and for 4 hrs (H). The RITC-labelled ZZ-BNC was used and the cells were fixed and permeabilized. EC-Fc or Fc were detected with anti-human IgG labelled with FITC (G) and ErbB2 was detected with sc-08 antibody followed by rabbit anti-mouse IgG Alexa 488 (H). Bars, 10 μm.
Mentions: The multivalent form of EC-1 peptide was prepared exploiting the affinity of ZZ-BNC for IgG Fc region (Fig. 3A). When ZZ-BNC was mixed with EC-Fc, the ligand EC-1 was multivalently displayed on the surface of ZZ-BNC (EC-Fc/BNC). On the other hand, ZZ-BNC displaying ligand was just prepared by mixing Fc protein with ZZ- BNC (Fc/BNC). First of all, binding capacity and binding efficiency of the ZZ-BNC with ligand was optimized and characterized. To optimize the ratio of Fc fusion molecule to BNC, FITC-labelled ZZ-BNC was mixed with Fc protein at variable molar ratios of 1:10, 1:20, 1:40, 1:60, 1:80 and 1:100, respectively, and the residual fluorescent intensity in the supernatant was measured. As shown in Figure 3B, the soluble Fc/BNC was reduced to 30% at a molar ratio of ZZ-BNC to Fc protein at 1:100 when judged from the fluorescence in the supernatant. Similarly, when human IgG was used, the soluble IgG/BNC was found reduced to 10% at a molar ratio of ZZ-BNC to IgG protein at 1:100. The amount of Fc protein bound to ZZ-BNC in the supernatant was further estimated from intensity of the band detected by Western blotting (Fig. 3C). As the result, the amount of Fc protein bound to ZZ-BNC was determined maximum when the molar ratio of ZZ-BNC to Fc protein was at 1:20. Thus, EC-Fc/ZZ-BNC was prepared by mixing ZZ-BNC and EC-Fc at the molar ratio of 1:20 for further experiments.

Bottom Line: Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand.Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells.Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nano-Biotechnology, Department of Medical Bioengineering Science, Graduate School of Natural Science and Biotechnology, Okayama University, Kita-ku, Okayama, Japan.

Show MeSH
Related in: MedlinePlus