title: Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin.
abstract: Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C-domain (251-370 residues; CP251- 370) in biological activities of the toxin. The N-domain (1-250 residues; CP1- 250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC-CP251-370) consisting of BcPLC and CP251- 370 bound to the red cells and lysed them. Incubation of CP1-250 with CP251-370 completely complemented hemolytic and PLC activities. CP251-370 also conferred hemolytic activity on BcPLC. CP251-340 (251-340 residues) significantly stimulated PLC activity of CP1-250), but did not confer hemolytic activity on CP1-250. Kinetic analysis suggested that CP251-370 increased affinity toward the substrate of CP1-250. The results suggested that CP251-370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1-250. Acrylodan-labeled CP251-370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251-370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251-370 to a hydrophobic environment. These observations suggest that interaction of CP251-370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1-250.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Tokushima 770-8514, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.forename: Masakazu
authors.initials: M
authors.lastname: Mukai
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Morimitsu
authors.forename: Sadayuki
authors.initials: S
authors.lastname: Ochi
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins; D003016:Clostridium perfringens / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism; D006461:Hemolysis / Q000502:physiology; D007700:Kinetics; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D010738:Type C Phospholipases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D008081:Liposomes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens