Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O ( θ-toxin), a c...

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Bibliographic Details
Published in:Anaerobe Vol. 10; no. 2; pp. 125 - 134
Main Authors: Ohno-Iwashita, Yoshiko, Shimada, Yukiko, Waheed, A.Abdul, Hayashi, Masami, Inomata, Mitsushi, Nakamura, Megumi, Maruya, Mikako, Iwashita, Shintaro
Format: Journal Article
Language:English
Published: England Elsevier Ltd 01-04-2004
Subjects:
Cholesterol-binding probe
Domain structure
Lipid rafts
Perfringolysin O
Visualization
Visualization
Cholesterol-binding probe
Lipid rafts
Domain structure
Perfringolysin O
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Summary:Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O ( θ-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BC θ) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BC θ can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BC θ, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BC θ. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.
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ISSN:1075-9964
1095-8274
DOI:10.1016/j.anaerobe.2003.09.003