Cold-labile hemolysin produced by limited proteolysis of .theta.-toxin from Clostridium perfringens

Ohno‐Iwashita, Yoshiko; Iwamoto, Machiko; Mitsui, Ken'ichiro; Kawasaki, Hiroshi; Ando, Susumu

doi:10.1021/bi00368a032

Cited by 46 publications

(50 citation statements)

References 26 publications

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“…-Toxin was overexpressed in Escherichia coli and purified from the periplasmic fractions as described (15). A nicked -toxin (C) was obtained by using subtilisin Carlsberg digestion (12) and BC was prepared as described (11).…”

Section: Methodsmentioning

confidence: 99%

“…On BC binding a shift in the distribution patterns of lipids and proteins was observed from fractions 2-4 to fractions 3-5 (ϩTx). Because Tx in bottom fractions (7)(8)(9)(10)(11)(12) interferes with the separation of lipids on TLC plates, the distribution pattern of sphingomyelin in these fractions was not shown (b, ϩTx).…”

Section: Methodsmentioning

confidence: 99%

“…-Toxin (perfringolysin O), a thiol-activated cytolysin produced by Clostridium perfringens, binds to membrane cholesterol (11)(12)(13)(14)(15)(16)(17)(18), and then forms oligomeric pores causing membrane damage (18,19). The C-terminal amino acid residues are essential for its activity (15,18).…”

mentioning

confidence: 99%

“…The three-dimensional structure of -toxin has been shown recently (19). The digested -toxin with subtilisin Carlsberg protease (C) is a complex of 38-and 15-KDa fragments and loses the capacity of oligomerization, resulting in no hemolytic activity below 20°C (12,13). Biotinylated C (BC) (11) has the same binding affinity to membrane cholesterol as wild-type -toxin and C. BC possesses no Abbreviations: -toxin, perfringolysin O; C, -toxin nicked with subtilisin Carlsberg protease; BC, biotinylated C; Tx, Triton X-100; FLDF, floating low-density fractions; M␤CD, methyl-␤-cyclodextrin; 2OHp␤CD, 2-hydroxypropyl-␤-cyclodextrin; IEM, immunoelectron microscopy.…”

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confidence: 99%

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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.

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…Each derivative is composed of two fragments: a basic N-terminal fragment (Tl, M , 28000; C2, M , 15000) and a weakly acidic Cterminal fragment (T2, M , 25 000; C1, M , 38 000) that contains the cysteine residue ( Fig. 1) [15]. Both fragments exist in complex form and have hemolytic activity.…”

Section: Andtoxin (Perfringolysin 0) Is a Cytolytic Toxin Secreted Bymentioning

confidence: 99%

Effect of isolated C‐terminal fragment of θ‐toxin (perfringolysin O) on toxin assembly and membrane lysis

Iwamoto

Ohno‐Iwashita

Ando

1990

European Journal of Biochemistry

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θ‐toxin, a thiol‐activated cytolysin, binds cholesterol and assembles on plasma membrane during the lytic process. In order to understand the process at the molecular level, two fragments (T1 and T2) were isolated from a nicked toxin obtained by limited proteolysis with trypsin. Although neither the T1 nor T2 fragment has hemolytic activity, T2 has almost the same potential as native θ‐toxin in its binding affinity for erythrocytes and in its binding specificity for cholesterol. T2, derived from the C‐terminus of the toxin, loses binding activity upon 5,5′‐dithiobis (2‐nitrobenzoic acid) modification of the thiol group. The T2 fragment was found to abolish the hemolytic activity of θ‐toxin completely without any inhibition of θ‐toxin binding to erythrocytes. θ‐toxin normally appears in polymeric form on membranes, while it remains in monomer form in the presence of the T2 fragment, as judged by sedimentation patterns in sucrose density‐gradient centrifugation. These results indicate that without inhibiting binding, the T2 fragment inhibits hemolysis by preventing θ‐toxin from aggregating on membranes, a step that might be essential for the lytic process.

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Real‐time monitoring of the membrane‐binding and insertion properties of the cholesterol‐dependent cytolysin anthrolysin O from Bacillus anthracis

Simon

Jost

Robertson

et al. 2006

J of Molecular Recognition

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Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.

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Cold-labile hemolysin produced by limited proteolysis of .theta.-toxin from Clostridium perfringens

Cited by 46 publications

References 26 publications

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

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

Effect of isolated C‐terminal fragment of θ‐toxin (perfringolysin O) on toxin assembly and membrane lysis

Real‐time monitoring of the membrane‐binding and insertion properties of the cholesterol‐dependent cytolysin anthrolysin O from Bacillus anthracis

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