title: Development of a screening method to identify regulators of MICA shedding.
abstract: Immune cells, such as natural killer (NK) cells, recognize virally infected and transformed cells, and eliminate them through the interaction between NKG2D receptors on NK cells and NKG2D ligands on pathogenic cells. Shedding of NKG2D ligands is thought to be a type of counter-mechanism employed by pathogenic cells to evade from NKG2D-mediated immune surveillance. MHC class I polypeptide-related sequence A (MICA) is a prototypical NKG2D ligand. We previously reported that, in soluble form, MICA expression levels are significantly associated with hepatitis virus-induced hepatocellular carcinoma. Here, we report a MICA shedding assay that utilizes membrane-bound MICA tagged at its N-terminus with a nano-luciferase reporter to quantify MICA shedding into culture media. Using this method, we screened a compound library and identified putative regulators of MICA shedding that have the potential to enhance the immune reaction by simultaneously increasing cell surface MICA levels and decreasing soluble MICA levels. This shedding assay may be useful for screening regulators of cell surface molecule shedding.
authors: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors: Takahiro
authors: T
authors: Kishikawa
authors: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan; Japan Science and Technology Agency, PRESTO, Kawaguchi, Saitama 332-0012, Japan. Electronic address: otsukamo-tky@umin.ac.jp.
authors: Motoyuki
authors: M
authors: Otsuka
authors: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors: Motoko
authors: M
authors: Ohno
authors: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors: Takeshi
authors: T
authors: Yoshikawa
authors: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors: Masaya
authors: M
authors: Sato
authors: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.
authors: Kazuhiko
authors: K
authors: Koike
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002472:Cell Transformation, Viral / Q000235:genetics / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics / Q000472:pathogenicity; D057166:High-Throughput Screening Assays / Q000379:methods*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008711:Metergoline / Q000494:pharmacology; D000078792:Midkine; D008969:Molecular Sequence Data; D008981:Molsidomine / Q000494:pharmacology; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D009414:Nerve Growth Factors / Q000276:immunology; D012995:Solubility
chemical_list: D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; C527085:KLRK1 protein, human; D008024:Ligands; C066254:MDK protein, human; C099187:MHC class I-related chain A; D055655:NK Cell Lectin-Like Receptor Subfamily K; D009414:Nerve Growth Factors; D000078792:Midkine; D008711:Metergoline; D008981:Molsidomine
keywords: Compound screening; MICA; Reporter; Shedding assay