Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of ratvascularsmoothmuscle

Sekizawa, Yoshiyuki; Kubo, Takeo; Kobayashi, Hideshi; Nakajima, Terumi; Natori, Shunji

doi:10.1016/s0378-1119(97)00047-4

Cited by 89 publications

(66 citation statements)

References 16 publications

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“…The earthworm E. foetida produces at least two lysenin-like proteins in addition to lysenin itself, and these three proteins have a high amino acid sequence identity (9,23). Another research group has recently found that a cytolysin (named eiseniapore) purified from the earthworm E. foetida recognizes SM (24).…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, another research group suggested that mammalian SPT did not require expression of an LCB1 protein, based on observations that overexpression of mouse LCB2 cDNA in human embryonic kidney 293 cells enhanced SPT activity in the cells even without co-expression of mouse LCB1 cDNA (6). It is thus currently controversial whether the SPT enzyme comprises two different subunits, namely the LCB1 and LCB2 proteins.Lysenin, a 41-kDa protein derived from the coelomic fluid of the earthworm Eisenia foetida, induces hemolysis of mammalian red cells (8,9). Recently, it has been shown that lysenin specifically binds to SM among various lipid types in cell-free systems (10).…”

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confidence: 99%

“…Lysenin, a 41-kDa protein derived from the coelomic fluid of the earthworm Eisenia foetida, induces hemolysis of mammalian red cells (8,9). Recently, it has been shown that lysenin specifically binds to SM among various lipid types in cell-free systems (10).…”

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confidence: 99%

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Mammalian Cell Mutants Resistant to a Sphingomyelin-directed Cytolysin

Hanada¹,

Hara²,

Fukasawa³

et al. 1998

Journal of Biological Chemistry

176

123

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Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin, but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lyseninresistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wildtype cells. One mutant strain was severely defective in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable of de novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface sphingomyelin is essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient mutants. Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.Sphingolipids are ubiquitous constituents of biomembranes in mammalian cells. The most abundant species of sphingolipid in mammalian cells is sphingomyelin (SM), 1 which amounts to 5-20% of total phospholipids. Sphingolipid biosynthesis is initiated by condensation of L-serine with palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT; EC 2.3.1.50) to generate 3-ketodihydrosphingosine (see Ref.1 for a review of sphingolipid biosynthesis). 3-Ketodihydrosphingosine is converted to dihydrosphingosine, which is N-acylated and then dehydrogenated to form ceramide at the endoplasmic reticulum. After moving to the Golgi apparatus, ceramide is converted to sphingomyelin or glycosphingolipids, and these synthesized complex sphingolipids are translocated to the plasma membrane, where they are highly enriched. SPT is suggested to be a key enzyme for regulation of cellular sphingolipid content (1). Regulation of sphingolipid synthesis at the SPT step appears to be relevant to prevention of a harmful accumulation of metabolic sphingolipid intermediates including sphingoid bases and ceramide, since repression of other anabolic steps in the sphingolipid synthetic pathway may cause the intermediate accumulation. Genetic studies have shown that two different genes, LCB1 and LCB2, are required for expression of SPT activity in the yea...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Mammalian Cell Mutants Resistant to a Sphingomyelin-directed Cytolysin

Hanada¹,

Hara²,

Fukasawa³

et al. 1998

Journal of Biological Chemistry

176

123

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“…Coelomic fluid (CF) and lysenin were prepared from the earthworm Eisenia foetida in our laboratory, as described previously (23,24). In most experiments, CF, in which the concentration of lysenin was 290 μg/mL (23), was used because of the limited amount of lysenin available to us.…”

Section: Methodsmentioning

confidence: 99%

“…Lysenin is a novel bioactive 33-kDa protein which we isolated from the coelomic fluid (CF) of the earthworm Eisenia foetida and it does not contain any amino acid sequences homologous to those of proteins in standard databases (13,23,24). This protein binds specifically to sphingomyelin (SM) among the various phospholipids found in the cell membrane (29).…”

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Exfoliation of the Epidermal cells and Defecation by Amphibian Larvae in Response to Coelomic Fluid and Lysenin from the Earthworm Eisenia foetida

2006

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Coelomic fluid (CF) and lysenin from the earthworm Eisenia foetida induced heavy epidermal exfoliation in the larvae of Bufo japonicus formosus at developmental stages from hatching (stage 22) to operculum completion (stage 34). In experiments with Xenopus laevis, we observed that exfoliated cells were not stained by trypan blue. Thus, it appeared that these cells were still alive. It is likely, therefore, that both CF and lysenin might disrupt the adhesion between epidermal cells of larvae prior to stage 34. Since it is known that lysenin exerts its toxic effects through its specific binding to sphingomyelin (SM), SM might be involved in such adhesion. This hypothesis was supported by the observations that CF and lysenin which had been incubated with SM-liposomes lost their exfoliative activity. In larvae after stage 34, the mechanism of adhesion between epidermal cells seemed to change and the adhesion was no longer disrupted by CF and lysenin. In larvae at around stage 34, a collagen layer started to form beneath the basement membrane of the epidermis. Furthermore, larvae at around this stage started to eat solid food. The developing collagen layer and food intake might be related indirectly to the chemical change in epidermal adhesion. The induction of exfoliation by CF and lysenin was also observed in other amphibian species. In Bufo larvae, defecation was induced both by CF and by lysenin but this effect was independent of exfoliation.

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Cell adhesion and the immune system: a case study using earthworms

Cooper

Cossarizza

Kauschke

et al. 1999

Microsc. Res. Tech.

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In the earthworm's immune system, cell adhesion, which occurs by putative receptors on leukocytes, is essential after recognition of self vs. non‐self. Confrontation with foreign antigens is a normal event in the environment, replete with microbial pathogens that pose a threat to survival. To better understand what happens when an effector cell first recognizes a foreign target followed by its adhesion to it, isolated leukocytes, in sufficient quantities to be subjected to various analyses, have been extremely beneficial. In vitro approaches when accompanied by biochemical, immunological, and molecular technologies, have opened up new vistas concerning the immune response of earthworms and other invertebrates. The most recent discovery includes the preliminary identification of cell differentiation (CD) markers that play vital roles in recognitive and adhesive events. Certain leukocyte effectors show characteristics of natural killer (NK) cells that may act differently depending upon their source, whether autogeneic, allogeneic, xenogeneic, or expressed under normal or varying environmental conditions including exposure to xenobiotics. At the level of earthworm evolution, there is apparently a dissociation of phagocytosis from the process of killing by NK‐like effectors. There are at least three future challenges. First, it is essential to determine the precise nature of the CD markers with respect to their molecular structure. Second, once their molecular and biochemical characteristics have been defined, the role of these markers in cellular and humoral mechanisms must be clarified in order to define effector cell products and resulting immune responses. Third, there is a need to differentiate between the several lytic factors that have been found in earthworms with respect to molecular structure, and biochemical and functional characterization. Microsc. Res. Tech. 44:237–253, 1999. © 1999 Wiley‐Liss, Inc.

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Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of ratvascularsmoothmuscle

Cited by 89 publications

References 16 publications

Mammalian Cell Mutants Resistant to a Sphingomyelin-directed Cytolysin

Mammalian Cell Mutants Resistant to a Sphingomyelin-directed Cytolysin

Exfoliation of the Epidermal cells and Defecation by Amphibian Larvae in Response to Coelomic Fluid and Lysenin from the Earthworm Eisenia foetida

Cell adhesion and the immune system: a case study using earthworms

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