Oligomerization and Pore Formation of a Sphingomyelin-specific Toxin, Lysenin

Yamaji‐Hasegawa, Akiko; Makino, Asami; Baba, Toru; Senoh, Yukiko; Kimura-Suda, Hiromi; Sato, Satoshi B.; Terada, Nobuo; Ohno, Shinichi; Kiyokawa, Etsuko; Umeda, Masato; Kobayashi, Toshihide

doi:10.1074/jbc.m213209200

Cited by 121 publications

(156 citation statements)

References 37 publications

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“…For example, cholesterol (Chol) and sphingomyelin (SM) are two known specific receptors for pore-forming bacterial toxins. 39,40 Therefore, we were interested in knowing whether a lipid receptor such as Chol or SM is required for toxinmembrane interaction of Cry1Ab. Cry1Ab oligomer complexes were incorporated equally into liposomes composed of different lipid molecules, with or without Chol and SM ( Figure 3f).…”

Section: Resultsmentioning

confidence: 99%

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

et al. 2005

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The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxinreceptor interactions in cytotoxicity, we established a cellbased system utilizing High Five insect cells stably expressing BT-R 1 , the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R 1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R 1 , which apparently activates a Mg 2 þ -dependent cellular signaling pathway.

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Section: Resultsmentioning

confidence: 99%

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

et al. 2005

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“…For detection of SM localized at the plasma membrane, the cells were stained on ice for 30 min with non-toxic lysenin fused to maltose-binding protein (MBP-lysenin) (25), kindly provided by Dr. T. Kobayashi (The Institute of Physical and Chemical Research (RIKEN), Japan). The cells were washed with ice-cold phosphate-buffered saline supplemented with 1% FCS and 0.1% NaN 3 and incubated with rabbit anti-MBP antiserum (New England BioLabs, Beverly, MA) on ice for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Expression Cloning of a Human cDNA Restoring Sphingomyelin Synthesis and Cell Growth in Sphingomyelin Synthase-defective Lymphoid Cells

Yamaoka

Miyaji

Kitano

et al. 2004

Journal of Biological Chemistry

208

178

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Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(؊) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(؊) cells were also highly susceptible to methyl-␤-cyclodextrin (M␤CD) as compared with the WR19L/Fas-SM(؉) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(؊) cells and M␤CD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile ␣ motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(؊) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against M␤CD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/ Fas-SM(؊) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(؊) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.Diverse kinds of phospho-and glycerolipids such as diacylglycerol (DAG), 1 inositol phosphatides, and phosphatidic acid are recognized as bioactive molecules in cell growth and survival (1, 2). Sphingolipid ceramide has recently emerged as a signal mediator of cell functions including apoptosis, differentiation, and secretion (3). Various stresses such as ultraviolet, irradiation, heat shock, hypoxia, and biological factors such as tumor necrosis factor-␣, interferon-␥, and Fas antibody require ceramide generation to execute apoptosis, suggesting the implications of SM as a source of ceramide generation in the induction of cell death (4, 5). It was reported that SM dose-dependently inhibits both deoxycholate-induced apoptosis and subsequent hyper-proliferation in colon epithelial cells (6) and decreases the number of aberrant crypts of colon (7), suggesting the implications of SM in cell death and growth.SM is produced by SM synthase, which is thought to be the only enzyme to synthesize SM in mammalian cells (8). The enzyme catalyzes the reaction in which phosphocholine moiety is transferred from phosphatidylcholine (PC) to ceramide. Thus, the activation of SM synthase subsequently increases the levels of DAG and decreases ceramide at the same time (8). DAG is an important signaling molecule for cell growth through protein kinase C activation (9 -12) and acts competitively against ceramide-induced a...

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“…To answer this question, we focused our attention on lysenin, a 297 amino acid PFT extracted from Eisenia foetida, which inserts hexameric pores (∼3 nm diameter) in artificial and natural lipid membranes containing sphingomyelin (SM) [41][42][43][44][45]. Several remarkable features make lysenin an excellent candidate for such studies.…”

Section: Introductionmentioning

confidence: 99%

“…Several remarkable features make lysenin an excellent candidate for such studies. Lysenin's cytolytic and hemolytic activity has been extensively studied [45,46], and its capability to tamper with the barrier function of artificial lipid bilayers is well-documented [42,43,47,48]. The complete structure of the oligomeric pore inserted into membranes is not yet resolved; however, relatively recent structural data of lysenin interacting with SM in a pre-pore state indicates the existence of a positively charged domain [49] which may promote specific electrostatic interactions with negatively charged adenosine phosphates, similar to the ionotropic P2X receptors [19,50,51].…”

Section: Introductionmentioning

confidence: 99%

Purinergic control of lysenin’s transport and voltage-gating properties

Bryant

Shrestha

Carnig

et al. 2016

Purinergic Signalling

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Lysenin, a pore-forming protein extracted from the coelomic fluid of the earthworm Eisenia foetida, manifests cytolytic activity by inserting large conductance pores in host membranes containing sphingomyelin. In the present study, we found that adenosine phosphates control the biological activity of lysenin channels inserted into planar lipid membranes with respect to their macroscopic conductance and voltage-induced gating. Addition of ATP, ADP, or AMP decreased the macroscopic conductance of lysenin channels in a concentration-dependent manner, with ATP being the most potent inhibitor and AMP the least. ATP removal from the bulk solutions by buffer exchange quickly reinstated the macroscopic conductance and demonstrated reversibility. Singlechannel experiments pointed to an inhibition mechanism that most probably relies on electrostatic binding and partial occlusion of the channel-conducting pathway, rather than ligand gating induced by the highly charged phosphates. The Hill analysis of the changes in macroscopic conduction as a function of the inhibitor concentration suggested cooperative binding as descriptive of the inhibition process. Ionic screening significantly reduced the ATP inhibitory efficacy, in support of the electrostatic binding hypothesis. In addition to conductance modulation, purinergic control over the biological activity of lysenin channels has also been observed to manifest as changes of the voltage-induced gating profile. Our analysis strongly suggests that not only the inhibitor's charge but also its ability to adopt a folded conformation may explain the differences in the observed influence of ATP, ADP, and AMP on lysenin's biological activity.

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Oligomerization and Pore Formation of a Sphingomyelin-specific Toxin, Lysenin

Cited by 121 publications

References 37 publications

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

Expression Cloning of a Human cDNA Restoring Sphingomyelin Synthesis and Cell Growth in Sphingomyelin Synthase-defective Lymphoid Cells

Purinergic control of lysenin’s transport and voltage-gating properties

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