title: Expressed glycosylphosphatidylinositol-anchored horseradish peroxidase identifies co-clustering molecules in individual lipid raft domains.
abstract: Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains.
authors.affiliation: Kochi System Glycobiology Center, Kochi University Medical School, Nankoku, Kochi, Japan; Center for Innovate and Translational Medicine, Kochi University Medical School, Nankoku, Kochi, Japan.
authors.forename: Arisa
authors.initials: A
authors.lastname: Miyagawa-Yamaguchi
authors.affiliation: Kochi System Glycobiology Center, Kochi University Medical School, Nankoku, Kochi, Japan; Center for Innovate and Translational Medicine, Kochi University Medical School, Nankoku, Kochi, Japan; Department of Biochemistry, Saitama Medical University, Iruma-gun, Saitama, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kotani
authors.affiliation: Kochi System Glycobiology Center, Kochi University Medical School, Nankoku, Kochi, Japan; Center for Innovate and Translational Medicine, Kochi University Medical School, Nankoku, Kochi, Japan; Department of Biochemistry, Kochi University Medical School, Nankoku, Kochi, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Honke
mesh_terms: D018958:CD55 Antigens / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018800:Thy-1 Antigens / Q000378:metabolism
chemical_list: D018958:CD55 Antigens; D005609:Free Radicals; D058851:GPI-Linked Proteins; D011993:Recombinant Fusion Proteins; D018800:Thy-1 Antigens; D006735:Horseradish Peroxidase