title: New mutant Chinese hamster ovary cell representing an unknown gene for attachment of glycosylphosphatidylinositol to proteins.
abstract: Aerolysin, a secreted bacterial toxin from Aeromonas hydrophila, binds to glycosylphosphatidylinositol (GPI)-anchored protein and kills the cells by forming pores. Both GPI and N-glycan moieties of GPI-anchored proteins are involved in efficient binding of aerolysin. We isolated various Chinese hamster ovary (CHO) mutant cells resistant to aerolysin. Among them, CHOPA41.3 mutant cells showed several-fold decreased expression of GPI-anchored proteins. After transfection of N-acetylglucosamine transferase I (GnT1) cDNA, aerolysin was efficiently bound to the cells, indicating that the resistance against aerolysin in this cells was mainly ascribed to the defect of N-glycan maturation. CHOPA41.3 cells also accumulated GPI intermediates lacking ethanolamine phosphate modification on the first mannose. After stable transfection of PIG-N cDNA encoding GPI-ethanolamine phosphate transferase1, a profile of accumulated GPI intermediates became similar to that of GPI transamidase mutant cells. It indicated, therefore, that CHOPA41.3 cells are defective in GnT1, ethanolamine phosphate modification of the first mannose, and attachment of GPI to proteins. The GPI accumulation in CHOPA41.3 cells carrying PIG-N cDNA was not normalized after transfection with cDNAs of all known components in GPI transamidase complex. Microsomes from CHOPA41.3 cells had normal GPI transamidase activity. Taken together, there is an unknown gene required for efficient attachment of GPI to proteins.
authors.affiliation: Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School, Gwangju, Republic of Korea. yjhong@chonnam.ac.kr
authors.forename: Yeongjin
authors.initials: Y
authors.lastname: Hong
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Kang
authors.forename: Youn Uck
authors.initials: YU
authors.lastname: Kim
authors.forename: Dong-Jun
authors.initials: DJ
authors.lastname: Shin
authors.forename: Hyon E
authors.initials: HE
authors.lastname: Choy
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Maeda
authors.forename: Taroh
authors.initials: T
authors.lastname: Kinoshita
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000008:administration & dosage* / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010770:Phosphotransferases; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C032676:aerolysin; D017351:N-Acetylglucosaminyltransferases; D010770:Phosphotransferases; C403312:Pign protein, mouse