Use of Clostridium septicum alpha toxins for isolation of various glycosylphosphatidylinositol-deficient cells.
In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.
Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School, Gwangju, Republic of Korea, 501-746.
Dong-Jun
DJ
Shin
Hyon E
HE
Choy
Yeongjin
Y
Hong
D000217:Acyltransferases / Q000235:genetics*; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology*; D016466:CHO Cells / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism*; D008364:Mannosyltransferases / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D052899:Pore Forming Cytotoxic Proteins; D014162:Transfection
D001427:Bacterial Toxins; D017261:Glycosylphosphatidylinositols; D052899:Pore Forming Cytotoxic Proteins; C064270:hemolytic toxin, Clostridium septicum; D007294:Inositol; C032676:aerolysin; D000217:Acyltransferases; D008364:Mannosyltransferases