[논문]Use of Clostridium septicum Alpha Toxins for Isolation of Various Glycosylphosphatidylinositol-Deficient Cells

Shin Dong-Jun; Choy Hyon E.; Hong Yeongjin

Use of Clostridium septicum Alpha Toxins for Isolation of Various Glycosylphosphatidylinositol-Deficient Cells 원문보기

The journal of microbiology, v.43 no.3, 2005년, pp.266 - 271

Shin Dong-Jun (Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School) , Choy Hyon E. (Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School) , Hong Yeongjin (Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School)

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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.

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All cDNA used in this work were constructed in a mammalian expression vector, pME (gifts from Dr. Taroh Kinoshita, Osaka University, Japan).
Finally, to investigate the expression of GPI-anchored proteins on the outer cell membrane of mutants, we stained cells with antibodies against GPI-anchored pro teins; DAF and CD59 (Fig. 2B). Wild-type D9(wt) cells showed high expression of GPI-anchored proteins.

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Use of Clostridium septicum Alpha Toxins for Isolation of Various Glycosylphosphatidylinositol-Deficient Cells 원문보기

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Use of Clostridium septicum Alpha Toxins for Isolation of Various Glycosylphosphatidylinositol-Deficient Cells 원문보기

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