Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria.
Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.
Shin
Dong-Jun
DJ
Department of Microbiology, Genomic Research Center for Enteropathogenic Bacteria, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea.
Lee
Je-Jung
JJ
Choy
Hyon E
HE
Hong
Yeongjin
Y
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D018958:CD55 Antigens / Q000096:biosynthesis; D018957:CD59 Antigens / Q000096:biosynthesis; D016466:CHO Cells; D003013:Clostridium / Q000378:metabolism*; D006224:Cricetinae; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006031:Glycosylation; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis* / Q000378:metabolism; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry; D052899:Pore Forming Cytotoxic Proteins
D001427:Bacterial Toxins; D018958:CD55 Antigens; D018957:CD59 Antigens; D011134:Polysaccharides; D052899:Pore Forming Cytotoxic Proteins; C064270:hemolytic toxin, Clostridium septicum; C032676:aerolysin; D005982:Glutathione Transferase