Effect of isolated C-terminal fragment of theta-toxin (perfringolysin O) on toxin assembly and membrane lysis.
theta-toxin, a thiol-activated cytolysin, binds cholesterol and assembles on plasma membrane during the lytic process. In order to understand the process at the molecular level, two fragments (T1 and T2) were isolated from a nicked toxin obtained by limited proteolysis with trypsin. Although neither the T1 nor T2 fragment has hemolytic activity. T2 has almost the same potential as native theta-toxin in its binding affinity for erythrocytes and in its binding specificity for cholesterol. T2, derived from the C-terminus of the toxin, loses binding activity upon 5,5'-dithiobis(2-nitrobenzoic acid) modification of the thiol group. The T2 fragment was found to abolish the hemolytic activity of theta-toxin completely without any inhibition of theta-toxin binding to erythrocytes. theta-toxin normally appears in polymeric form on membranes, while it remains in monomer form in the presence of the T2 fragment, as judged by sedimentation patterns in sucrose density-gradient centrifugation. These results indicate that without inhibiting binding, the T2 fragment inhibits hemolysis by preventing theta-toxin from aggregating on membranes, a step that might be essential for the lytic process.
Iwamoto
M
M
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Japan.
Ohno-Iwashita
Y
Y
Ando
S
S
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D003016:Clostridium perfringens; D003545:Cysteine / Q000502:physiology; D004910:Erythrocyte Membrane / Q000378:metabolism; D006460:Hemolysin Proteins; D006461:Hemolysis; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism / Q000633:toxicity; D012756:Sheep; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008563:Membrane Lipids; D010446:Peptide Fragments; C020452:Clostridium perfringens theta-toxin; D014357:Trypsin; D003545:Cysteine