A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.
We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.
Ohno-Iwashita
Y
Y
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Gerontology.
Iwamoto
M
M
Mitsui
K
K
Ando
S
S
Iwashita
S
S
D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D002784:Cholesterol / Q000378:metabolism*; D003016:Clostridium perfringens; D006460:Hemolysin Proteins / Q000378:metabolism; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D008854:Microscopy, Electron; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; D011485:Protein Binding; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol