title: Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody.
abstract: A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces.
authors: †Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors: Takahiro
authors: T
authors: Hayashi
authors: †Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors: Yuki
authors: Y
authors: Yasueda
authors: †Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors: Tomonori
authors: T
authors: Tamura
authors: †Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors: Yousuke
authors: Y
authors: Takaoka
authors: †Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.|‡Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
authors: Itaru
authors: I
authors: Hamachi
mesh_terms: D015761:4-Aminopyridine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D018604:Epitope Mapping / Q000379:methods*; D066246:ErbB Receptors / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D008958:Models, Molecular; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis*
chemical_list: D015761:4-Aminopyridine; D066246:ErbB Receptors; D018796:Immunoconjugates; D018719:Erb-b2 Receptor Tyrosine Kinases; D011956:Receptors, Cell Surface; C003885:4-dimethylaminopyridine