Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand.
Bottom Line: In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2.This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway.Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line.
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.