Reduced sensitivity of Niemann-Pick C1-deficient cells to theta-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles.
Theta-toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(-) that lacked Niemann-Pick C1 showed reduced sensitivity to theta-toxin, compared with wild-type (wt) cells. BCtheta is a derivative of theta-toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BCtheta, both on the cell surface and in the extracellular space of these cells. BCtheta binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4 degrees C and recovery of bound BCtheta in floating low-density fractions on sucrose density gradient fractionation. BCtheta-labeled vesicles were abolished when NPC1(-) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, theta-toxin sensitivity of NPC1(-) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to theta-toxin.
Ohsaki
Yuki
Y
Department of Neurobiology, Tottori University Faculty of Medicine, 683-8503, Yonago, Japan.
Sugimoto
Yuko
Y
Suzuki
Michitaka
M
Kaidoh
Toshiyuki
T
Shimada
Yukiko
Y
Ohno-Iwashita
Yoshiko
Y
Davies
Joanna P
JP
Ioannou
Yiannis A
YA
Ohno
Kousaku
K
Ninomiya
Haruaki
H
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism; D004351:Drug Resistance; D006460:Hemolysin Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000172:deficiency* / Q000235:genetics / Q000502:physiology*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008854:Microscopy, Electron; D000077296:Niemann-Pick C1 Protein; D011374:Progesterone / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D014162:Transfection; D054460:rho-Associated Kinases
D001427:Bacterial Toxins; D002352:Carrier Proteins; D006460:Hemolysin Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D011374:Progesterone; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases