title: Effective capture of proteins inside living cells by antibodies indirectly linked to a novel cell-penetrating polymer-modified protein A derivative.
abstract: Antibodies against cytoplasmic proteins are useful tools that can control cellular function and clarify signaling mechanisms. However, it is difficult to capture proteins inside living cells, and thus appropriate methods for antibody delivery to the cytoplasm of living cells are required. Cell-penetrating materials, such as the TAT-peptide, have received attention for their ability to deliver various cargos into living cells. However, the direct modification of cargos with cell-penetrating materials is time-consuming and lacks versatility. Therefore, we conceived that protein A, which can bind to the fragment crystallizable region of an antibody, could indirectly link antibodies with cell-penetrating materials, creating an efficient and simple antibody delivery system. Here, we constructed a novel antibody delivery system using a cell-penetrating polymer-modified protein A derivative (CPP-pAd). Living cells treated with CPP-pAd/antibody complexes showed significantly higher antibody levels than those achieved with the commercially available reagent HVJ-E. Pre-treatment with sucrose prevented cellular uptake of the CPP-pAd/antibody complex, suggesting that the CPP-pAd/antibody internalization mechanism occurs through clathrin-dependent endocytosis. Interestingly, intracellularly delivered antibodies did not colocalize with endosome/lysosome markers, further suggesting that antibodies were delivered to the cytoplasm by escape from endosome/lysosome. Moreover, we observed that anti-nuclear pore complex antibodies, delivered to cells using CPP-pAd, localized to the nuclear membrane and inhibited nuclear factor κB dependent luciferase activity. Together, these results suggest that the antibodies delivered by CPP-pAd captured functional proteins, making CPP-pAd a promising strategy for effective capture of proteins inside living cells.
authors.affiliation: Department of Biophysical Chemistry, Kyoto Pharmaceutical University, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Itakura
authors.forename: Susumu
authors.initials: S
authors.lastname: Hama
authors.forename: Hisafumi
authors.initials: H
authors.lastname: Ikeda
authors.forename: Naoto
authors.initials: N
authors.lastname: Mitsuhashi
authors.forename: Eiji
authors.initials: E
authors.lastname: Majima
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kogure
mesh_terms: D000906:Antibodies / Q000008:administration & dosage*; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage* / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D016503:Drug Delivery Systems; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology* / Q000378:metabolism*; D015394:Molecular Structure; D028861:Nuclear Pore Complex Proteins / Q000276:immunology / Q000378:metabolism; D011108:Polymers / Q000008:administration & dosage / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000008:administration & dosage* / Q000737:chemistry
chemical_list: D000906:Antibodies; D057846:Cell-Penetrating Peptides; D047908:Intracellular Signaling Peptides and Proteins; D028861:Nuclear Pore Complex Proteins; D011108:Polymers; D013205:Staphylococcal Protein A
keywords: antibody; cell-penetrating polymer; cytoplasmic proteins; endosomal escape; protein A