C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Shimada, Yukiko; Nakamura, Megumi; Naito, Yasuhide; Nomura, Kohji; Ohno‐Iwashita, Yoshiko

doi:10.1074/jbc.274.26.18536

Cited by 46 publications

(41 citation statements)

References 35 publications

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“…We identified two domains of VVA2 responsible for its membrane binding, oligomerization, and membrane insertion. In the cholesterol-dependent cytolysins family, it has been shown that the C-terminal domain is responsible for membrane attachment and contains the longest conserved tryptophan-rich sequence motif, a situation similar to equinatoxin II, a well known eukaryotic pore-forming toxin (12,32). Here, we used Western blot and SPR analyses to show that VVA2 binds to cell membranes by its CTF.…”

Section: Discussionmentioning

confidence: 99%

“…The ␣-hemolysin produced by Staphylococcus aureus is divided into three domains: the rim, cap, and stem responsible for its membrane binding, oligomerization, and insertion, respectively (11). Perfringolysin O, another member of the cholesterol-dependent cytolysins family, the function of membrane binding is assigned to domain 4, oligomerization to domain 1, and insertion to domain 3 (5,12,13).…”

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confidence: 99%

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Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Weng

Lin

Hsu

et al. 2004

Journal of Biological Chemistry

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Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic ␣-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128 -199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing ␤6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166 -194 residues), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion.Volvatoxin A (VVA), 1 first isolated from the edible mushroom Volvariella volvacea (1, 2), is a novel pore-forming cardiotoxic protein that causes cardiac arrest by activation of Ca 2ϩ -dependent ATPase activity and inhibition of Ca 2ϩ accumulation in the microsomal fraction of the sarcoplasmic recticulum of the ventricular muscle (3). VVA is composed of VVA1 and VVA2, of which only VVA2 is endowed with hemolytic and cytotoxic activity (1). Our previous studies showed that VVA2 consists of 199 amino acid residues without cysteine residues, and that it forms pores in response to its oligomer formation in human erythrocyte membranes and liposomes. This toxic protein also permeabilized the cell membrane and allowed the passage of 70-kDa dextran rhodamine B into cultured Xenopus myocytes. Furthermore, conformational changes occurred when VVA2 interacted with liposomes (2).Naturally occurring hemolytic toxic proteins can be classified into three groups based on their mechanisms of lysis of red blood cells: (a) pore forming, (b) enzymatic degradation of membrane lipids, and (c) solubilizing cell membrane by a detergentlike action (4). Several hemolytic toxic proteins isolated...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Weng

Lin

Hsu

et al. 2004

Journal of Biological Chemistry

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“…It has previously been shown for other toxins such as PFO, PLY and SLO that domain 4 functions as the cell membrane binding (4,33,35). Therefore, we characterized the binding ability of recombinant ILY domain 4 (ILY4D) to human and rabbit ghosts.…”

Section: Identification Of the Key Part For The Human-specific Actionmentioning

confidence: 99%

“…Subsequent studies using recombinant domain 4 from these toxins showed that the cholesterol-binding site lies within domain 4 (4,33,35). As a result of this shared characteristic of cholesterol binding no other cytolysins belonging to this gene family exhibit species specificity in their action.…”

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confidence: 99%

The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein

Nagamune

Ohkura

Sukeno

et al. 2004

Microbiology and Immunology

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Intermedilysin is a pore‐forming cytolysin belonging to the streptolysin O gene family known as the ‘Cholesterol‐binding/dependent cytolysins’ and is unique within the family in that it is highly human‐specific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome‐embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome‐binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human‐specific step of intermedilysin action, that the host cell membrane‐binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human‐specific action step and the cell‐binding site of domain 4 lies within the 56 amino acids of the C‐terminal, excluding the undecapeptide region.

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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).…”

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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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C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Cited by 46 publications

References 35 publications

Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein

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

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