Role of tyrosine-57 and -65 in membrane-damaging and sphingomyelinase activities of Clostridium perfringens alpha-toxin.
Clostridium perfringens alpha-toxin (370 residues) is a major virulence factor in the pathogenesis of gas gangrene. The toxin is composed of an N-terminal domain (1-250 residues) where lies the catalytic site and a C-terminal domain (251-370 residues), the Ca(2+)-binding domain, responsible for binding to membranes. The role of Tyr-57 and Tyr-65 close to the catalytic pocket (site) in the N-domain was investigated. Replacement of Tyr-57 and -65 with alanine, leucine, or phenylalanine did not affect the sphingomyelinase activity of the toxin for sodium deoxycholate-solubilized shingomyelin. However, the substitution of Tyr-57 and -65 with alanine or leucine resulted in a radical reduction in the hemolysis of sheep erythrocytes, the release of carboxyfluorescein from shingomyelin-cholesterol (1:1) liposomes, and a significant decrease in binding to the liposomes. The binding of variant toxins, Y57C/C169L and Y65C/C169L, labeled with the environmentally sensitive fluorophore, acrylodan, to the liposomes suggested insertion of the variants in a hydrophobic environment in the bilayer. These observations suggested that Tyr-57 and -65 play a role in the penetration of the toxin into the bilayer of membranes and access of the catalytic site to sphingomyelin in membranes, but do not participate in the enzymatic activity.
Nagahama
Masahiro
M
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan.
Otsuka
Akiko
A
Sakurai
Jun
J
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000473:pathology*; D002942:Circular Dichroism; D004912:Erythrocytes / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012756:Sheep; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics*
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D008081:Liposomes; D014443:Tyrosine; C038518:acrylodan; D015081:2-Naphthylamine; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; C040649:alpha toxin, Clostridium perfringens