Diagnosis of paroxysmal nocturnal hemoglobinuria by fluorescent clostridium septicum alpha toxin.
Paroxysmal nocturnal hemoglobinuria (PNH), a hematopoietic stem cell disorder, is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell membrane. PNH can be simply diagnosed by flow cytometry using monoclonal antibodies against GPI-anchored proteins or fluorescent-tagged aerolysin, a bacterial toxin that binds GPI anchored proteins. Clostridium septicum alpha toxin is homologous to aerolysin and specifically binds GPI-anchored proteins. Previously, we found that an alpha toxin m45 mutant with two amino acid changes, S189C/S238C, lost cytotoxicity but still possessed binding activity for GPI-anchored proteins. To use this mutant toxin as a diagnostic probe in flow cytometry, we constructed the EGFP-AT(m45) expression vector, comprising a S189C/S238C alpha toxin mutant with EGFP and His tags at the N and C termini, respectively. The recombinant EGFP-AT(m45) was easily purified using single-step affinity chromatography against His tag from Escherichia coli. EGFP-AT(m45) bound to CHO and HeLa cells in a similar manner to monoclonal antibodies against GPI-anchored proteins or aerolysin. In whole blood from a PNH patient, GPI-deficient granulocytes could be differentiated by EGFP-AT(m45) using the same procedure as that employed with commercially available monoclonal antibodies. Therefore, nontoxic EGFP-conjugated C. septicum alpha toxin could be used clinically for PNH diagnosis.
Shin
Dong-Jun
DJ
Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School, Gwangju, Korea.
Cho
Duck
D
Kim
Young Ran
YR
Rhee
Joon Haeng
JH
Choy
Hyon E
HE
Lee
Je-Jung
JJ
Hong
Yeongjin
Y
D000818:Animals; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins / Q000235:genetics*; D018958:CD55 Antigens / Q000276:immunology; D018957:CD59 Antigens / Q000276:immunology; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006098:Granulocytes / Q000378:metabolism / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006457:Hemoglobinuria, Paroxysmal / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D018958:CD55 Antigens; D018957:CD59 Antigens; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C064270:hemolytic toxin, Clostridium septicum; D049452:Green Fluorescent Proteins