A biotinylated perfringolysin O derivative: A new probe for detection of cell surface cholesterol

Iwamoto, Machiko; Morita, Ikuo; Fukuda, Mitsugu; Murota, Sei‐itsu; Ando, Susumu; Ohno‐Iwashita, Yoshiko

doi:10.1016/s0005-2736(97)00061-8

Cited by 58 publications

(69 citation statements)

References 41 publications

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“…Filipin complex from Streptomyces filipinensis (F9765) was purchased from Sigma-Aldrich (St. Louis, MO). BC 20 was provided by Dr. Yoshiko Ohno-Iwashita (Cellular Signaling Group, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan). High performance silica gel 60 thin layer chromatographic plates were from Merck (Darmstadt, Germany).…”

Section: Antibodies and Reagentsmentioning

confidence: 99%

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

Zhou

Davidson

McGlynn

et al. 2011

The American Journal of Pathology

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Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (␤1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently colocalized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal ␤-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol.

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Section: Antibodies and Reagentsmentioning

confidence: 99%

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

Zhou

Davidson

McGlynn

et al. 2011

The American Journal of Pathology

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“…2 and 3) based on several lines of evidence including one that CDCs are not cytolytically active on cholesterol-deficient membranes and another that the depletion of membrane cholesterol reduces the extent of membrane binding to various eukaryotic cell types (4). Many studies, primarily performed with perfringolysin O (PFO), have shown that membrane binding is sensitive to the loss of cholesterol (4)(5)(6)(7)(8)(9)(10)(11) and that derivatives of PFO have been used as probes for membrane cholesterol (12)(13)(14)(15).…”

mentioning

confidence: 99%

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins

Giddings

Johnson

Tweten

2003

Proc. Natl. Acad. Sci. U.S.A.

176

190

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The cholesterol-dependent cytolysins (CDCs) constitute a large family of pore-forming toxins that function exclusively on cholesterol-containing membranes. A detailed analysis of the various stages in the cytolytic mechanism of three members of the CDC family revealed that significant depletion of cholesterol from the erythrocyte membrane stalls these toxins in the prepore complex. Therefore, the depletion of membrane cholesterol prevents the insertion of the transmembrane ␤-barrel and pore formation. These unprecedented findings provide a paradigm for the involvement of cholesterol in the CDC cytolytic mechanism and that of other pore-forming toxins whose activity is enhanced by the presence of membrane cholesterol.

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“…To understand the role of rafts in various cellular functions, identification and analysis of the molecular organization of cholesterol in rafts are essential. Cholesterol-binding toxin would be a useful tool for such a purpose (11).…”

mentioning

confidence: 99%

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

Waheed

Shimada²,

Heijnen³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

207

182

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There is increasing evidence that sphingolipid-and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BC) of perfringolysin O (-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BC on a sucrose gradient. BC was predominantly localized in the floating lowdensity fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BC binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BC binding to FLDF. The staining patterns of BC and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BC binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BC binding does not cause any damage to cell membranes, indicating that BC is a useful probe for the detection of membrane rafts in living cells.

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A biotinylated perfringolysin O derivative: A new probe for detection of cell surface cholesterol

Cited by 58 publications

References 41 publications

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

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