Assembly and channel-forming activity of a naturally-occurring nicked molecule of Staphylococcus aureus alpha-toxin.
From the culture supernatant of Staphylococcus aureus Wood 46, we obtained a naturally-occurring nicked molecule of staphylococcal alpha-toxin. The nicked alpha-toxin consisted of non-covalently-linked 8-kDa and 25-kDa polypeptides, which were derived, respectively, from the N-terminal and the C-terminal part of the toxin nicked at the peptide bond between Glu-71 and Gly-72. The nicked toxin, as well as native alpha-toxin, bound to and oligomerized in the liposome membranes composed of choline-containing phospholipids (i.e., phosphatidylcholine and sphingomyelin) and cholesterol, and formed membrane channel in the liposome membranes. However, the channel-forming activity of the nicked toxin, assessed as a toxin-induced carboxyfluorescein leakage from the liposomes, was approx. 20-fold lower than that of native alpha-toxin. Channel-forming activity of the nicked toxin as well as native toxin was inhibited by divalent cations including Zn2+, Cd2+, Ca2+ and Mg2+, and degree of the inhibitory effect of the divalent cations was in the following order: Zn2+ > Cd2+ > Ca2+ > Mg2+. Thus, although the cleavage of alpha-toxin at the position between Glu-71 and Gly-72 drastically reduced channel-forming activity of the toxin, the nicked toxin retained the ability to oligomerize in phospholipid-cholesterol membranes and the characteristics of channel-forming activity in terms of the specificity for phospholipids and the susceptibility to divalent cations.
Tomita
T
T
Institute of Medical Science, University of Tokyo, Japan.
Watanabe
M
M
Yarita
Y
Y
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification; D002413:Cations, Divalent / Q000737:chemistry; D005452:Fluoresceins; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D013211:Staphylococcus aureus / Q000737:chemistry*
D001427:Bacterial Toxins; D002413:Cations, Divalent; D005452:Fluoresceins; D006460:Hemolysin Proteins; D008081:Liposomes; D010455:Peptides; D010743:Phospholipids; C021091:staphylococcal alpha-toxin; C024098:6-carboxyfluorescein