Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.
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.
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
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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.
Affiliation: Laboratory of Nano-Biotechnology, Department of Medical Bioengineering Science, Graduate School of Natural Science and Biotechnology, Okayama University, Kita-ku, Okayama, Japan.