Lysenin, a Novel Sphingomyelin-specific Binding Protein*

Akiko YamajiDagger §, Yoshiyuki Sekizawa, Kazuo EmotoDagger , Hitoshi Sakurabapar , Keizo Inoue§, Hideshi Kobayashi, and Masato UmedaDagger **

From the Dagger  Department of Inflammation Research and par  Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, the  Research Laboratory, Zenyaku Kogyo Co. Ltd., Nerimaku, Tokyo 178, and the § Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcoreTM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (kon = 3.2 × 104 M-1 s-1) and dissociated extremely slowly (koff = 1.7 × 10-4 s-1), giving a low dissociation constant (KD = 5.3 × 10-9 M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Sphingomyelin, N-acylsphingosine-1-phosphorylcholine, is a major lipid constituent of animal cell membranes, accounting for up to 60 mol % of the total phospholipid content of some cells, such as sheep erythrocytes (1). In addition to its role as a structural component of membranes, recent studies have suggested the roles of sphingomyelin in various intracellular signaling pathways (2-6). A sphingomyelin cycle has been proposed in which extracellular agents such as tumor necrosis factor alpha , gamma -interferon, and interleukin 1 activate sphingomyelinase, resulting in the production of ceramide which serves as a second messenger mediating the action of these extracellular ligands (4-6). Sphingomyelin was also proposed to be enriched in an organized membrane domain, caveolae, which contains cholesterol, glycosphingolipids, glycosylphosphatidylinositol-anchored proteins, and an integral membrane protein caveolin (7-11). Recently, glycosylphosphatidylinositol-anchored proteins and glycosphingolipids were reported to be sequestered in caveolae only after cross-linking of these molecules by antibodies, implying dynamic reorganization of the membrane microdomain structure during membrane signal transduction processes (12-14). Although these studies have suggested that sphingomyelin plays a role in the cellular signaling pathways and organization of the specialized membrane domains, little is known about where sphingomyelin is localized in membranes and how it participates in cellular functions, mainly due to a lack of appropriate methodologies for manipulating and tracing the functions of membrane phospholipids.

We have established various probes for studying the molecular motion of membrane phospholipids, including a small cyclic peptide that binds specifically to phosphatidylethanolamine (15, 16), and monoclonal antibodies against phosphatidylcholine (17), phosphatidylserine (18-20), and phosphatidylinositol 4,5-bisphosphate (21, 22). In this study, we demonstrated that a 41-kDa novel protein in the earthworm Eisenia foetida, designated lysenin (23, 24), which causes contraction of rat vascular smooth muscle, bound specifically to sphingomyelin. The specific binding of lysenin to sphingomyelin was further confirmed by ELISA,1 TLC immunostaining, liposome lysis assay, and kinetic analysis of the lysenin-sphingomyelin interaction using BIAcoreTM system, an optical biosensor based on the principles of surface plasmon resonance (25). The coelomic fluid of E. foetida has been found to exhibit various biological activities, such as hemolytic, antibacterial, hemagglutinating, and cytotoxic activities (23, 26, 27). A preliminary study by Roch et al. (28) showed that the hemolytic activity of the coelomic fluid of E. foetida was inhibited by vesicles containing sphingomyelin, and two proteins with molecular masses of 40 and 45 kDa could associate with the vesicles containing sphingomyelin and cholesterol. These components have not been isolated nor have their interactions with sphingomyelin been analyzed in detail. Recently Lange et al. (29) isolated a hemolysin, named as eiseniapore, from the coelomic fluid of E. fetida and showed that the protein bound to both sphingomyelin and galactosylceramide but not with ceramide or galactosylsphingosine, suggesting that eiseniapore recognizes the ceramide structure as well as a hydrophilic moiety of sphingolipids (30). In this study we provide the concrete evidence that lysenin binds specifically to sphingomyelin, and the immunocytochemical localization of sphingomyelin by lysenin in fibroblasts derived from a patient with Niemann-Pick disease type A suggested that lysenin should be a useful tool to probe the molecular motion and function of sphingomyelin in cellular membranes.

    EXPERIMENTAL PROCEDURES
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Purification of Lysenin and Polyclonal Antibody Preparation-- Lysenin from coelomic fluid of E. foetida was purified to a homogeneity by ammonium sulfate precipitation followed by sequential high performance column chromatography using TSKgel DEAE-2SW and TSKgel G2000SW (Tosoh Co., Tokyo), as described previously (23). The protein yielded a single band after SDS-polyacrylamide gel electrophoresis, and no contaminating protein bands were detected after staining the gel with Coomassie Brilliant Blue. An antiserum against lysenin was raised in male rabbits, as described previously (31), and its specificity was confirmed by immunoblotting analysis, which showed the antiserum recognized only one band of lysenin among all the proteins in the body fluid of E. foetida.

Measurement of Hemolysis-- Erythrocytes from various sources were washed and suspended in phosphate-buffered saline (PBS, 137 mM NaCl, 1.5 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4). Erythrocyte suspensions (1 ml containing 3 × 107 cells) were incubated with various concentrations of lysenin at 37 °C for 30 min and then centrifuged at 500 × g for 5 min to precipitate the erythrocytes. Aliquots of the supernatants were taken, and the optical densities at 415 nm were measured to determine the percentage of hemoglobin released from the erythrocytes. Total hemoglobin contents were determined by measuring hemoglobin released after freezing and thawing of the erythrocyte preparations. The binding of lysenin to various phospholipid vesicles was examined by performing a hemolysis inhibition assay, in which lysenin was preincubated with various phospholipid vesicles for 30 min at room temperature and then measuring the hemolytic activities of the lysenin-phospholipid vesicle mixtures against sheep erythrocytes, as described above. Phospholipid vesicles were prepared by sonication of multilamellar vesicles using a Branson Sonifier model 250D, as described previously (18). Phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphatidylinositol were prepared as described previously (18). Sphingomyelin, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine were purchased from Avanti Polar Lipids, and cholesterol was from Sigma.

ELISA-- Lysenin binding to various phospholipids was evaluated by ELISA, as described previously (18). In brief, the wells of microtiter plates (Immulon 1, Dynatech Laboratories, Alexandria, VA) were coated with 50 µl of phospholipid antigen (10 µM) in ethanol by evaporation at room temperature. After blocking the wells with Tris-buffered saline (TBS, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 30 mg/ml bovine serum albumin (BSA), the wells were incubated with various concentrations of lysenin in TBS containing 10 mg/ml BSA (1% BSA-TBS) for 2 h at room temperature. After washing the wells with TBS, the bound lysenin was detected by incubating the wells with anti-lysenin antiserum, diluted 1/1000 with 1% BSA-TBS for 2 h at room temperature, followed by incubation (2 h at room temperature) with biotinylated anti-rabbit IgG and peroxidase-conjugated streptavidin. The intensity of the color developed with o-phenylenediamine as the substrate was measured using an ELISA reader (MTP-32, Corona Electric, Tokyo).

TLC Immunostaining-- The TLC immunostaining assay was performed at room temperature, as described previously (32). Aliquots (10 and 50 nmol) of lipid antigen were spotted onto high performance TLC plates (5547, Merck Co., Darmstadt, Germany), which were dipped into a chloroform/hexane (1:100, v/v) solution containing 0.01% (w/v) polyisobutylmethacrylate (Aldrich) for 30 s, dried, soaked in PBS for 5 min, and then blocked with PBS containing 1% (w/v) BSA for 1 h. Then the plates were incubated with 1 µg/ml lysenin in 1% BSA-PBS for 2 h, washed with 1% BSA-PBS, incubated with anti-lysenin antiserum, diluted 1/1000 with 1% BSA-PBS, followed by biotinylated anti-rabbit IgG and peroxidase-conjugated streptavidin. Lysenin bound to the plate coated with lipids was detected using 4-chloro-1-naphthol solution (0.5 mg/ml) as the substrate. Ceramide and galactosylceramide were purchased from Avanti Polar Lipids, sphingosine and sphingosine 1-phosphate from Biomol Research Labs, and sphingosylphosphorylcholine from Matreya Inc.

Liposome Lysis Assay-- Multilamellar liposomes were prepared, and liposome lysis assay was performed as described previously (33) using calcein as a fluorescent marker (34). In brief, phospholipids were hydrated in 75 mM calcein (Sigma). Untrapped calcein was removed by centrifugation of the liposome suspension in PBS. Liposome lysis assay was performed in the wells of polyvinyl chloride microtiter plates (Immulon 1, Dynatech). The total reaction mixture (100 µl) containing 2 µM liposome suspension in PBS was incubated with various amounts of lysenin for 30 min at 37 °C, and the fluorescence of calcein was monitored using MTP-32 fluorescence microplate reader (Corona Electric, Tokyo) with excitation and emission wavelength at 490 and 530 nm, respectively. One hundred percent efflux of calcein was determined after freezing and thawing of the liposomes.

Kinetic Analysis of Lysenin Binding to Immobilized Phospholipid Membranes-- Lysenin binding to phospholipid membranes was quantified using a BIAcoreTM system instrument (Pharmacia Biosensor AB, Uppsala, Sweeden), an optical biosensor based on the principles of surface plasmon resonance (35). The sensor chip HPA has a surface composed of long chain alkane thiol molecules on a flat quasi-crystalline hydrophobic layer (Pharmacia Biosensor AB), and lipid monolayers were formed on the sensor chip by fusion of phospholipid vesicles with the alkane thiol monolayer by following the manufacturer's protocol. Small unilamellar vesicles composed entirely of sphingomyelin or a combination of either sphingomyelin and DPPC or sphingomyelin and cholesterol were prepared in PBS, as described above. To immobilize the phospholipid layer on the sensor chip, the chip was washed with 40 mM octyl glucoside, and vesicles containing 0.5 mM lipids were injected into the BIAcore system at a flow rate of 5 µl/min at 25 °C. Various concentrations of lysenin was injected over the immobilized membrane surface at a flow rate of 20 µl/min at 25 °C. The analyte binding assay was performed repeatedly, after washing the surface with 30 mM NaOH at a flow rate of 20 µl/min. The binding kinetics were analyzed according to the manual of the software BIAevaluation 2.1. The resonance unit is an arbitrary unit used by the BIAcore system, and there is a linear relationship between the mass of molecule bound to the sensor chip and the resonance unit observed (25).

Immunofluorescence Staining of Human Fibroblasts by Lysenin-- Skin fibroblasts from a patient with Niemann-Pick disease type A (NP-A) and normal subjects were established and maintained in our laboratory as described previously (36). The patient, a 1-year-old Japanese male, developed clinical manifestations including hepatosplenomegaly, hypotonia, muscular weakness, and psychomotor retardation. The diagnosis of NP-A was established by enzyme assaying of acid sphingomyelinase. Fibroblasts were cultured and maintained in Ham's F-10 medium containing 10% (v/v) fetal calf serum at 37 °C in a 5% CO2, 95% air incubator. The staining procedures were carried out at room temperature. Fibroblasts were incubated with 5 mg/ml fluorescein-labeled dextran (molecular mass 10,000, lysine fixable, Molecular Probes) for 24 h. After washing with PBS, the cells were fixed with 3.7% (w/v) formaldehyde in PBS, washed, and permeabilized with digitonin (50 µg/ml) for 10 min, blocked with 2% BSA-PBS for 15 min. The cells were incubated with lysenin (1 µg/ml in 2% BSA-PBS) for 2 h, washed with PBS, and incubated with anti-lysenin antiserum diluted 1/1000 with 2% BSA-TBS for 1 h, followed by incubation with 10 µg/ml tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit IgG (Chemicon International Inc., Temecula, CA) in 2% BSA-PBS for 1 h. Fluorescence microscopy was performed using a Zeiss Axioplan microscope.

    RESULTS AND DISCUSSION
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Introduction
Procedures
Results & Discussion
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Inhibition of Lysenin-induced Hemolysis by Sphingomyelin-- Lysenin caused hemolysis of human, rat, and sheep erythrocytes in a concentration-dependent manner (Fig. 1). The sensitivity to lysenin differed among animal species, and sheep erythrocytes were the most sensitive to lysenin, 5 ng/ml which was required to induce 50% hemolysis, a concentration 20 times lower than that required to hemolyze 50% of human and rat erythrocytes. During the course of our search for a cell-surface receptor for lysenin, we found that a total lipid extract from sheep erythrocyte membranes inhibited lysenin-induced hemolysis (data not shown). A hemolysis inhibition assay using synthetic lipid vesicles with various lipid compositions showed that vesicles containing sphingomyelin inhibited the hemolysis, whereas those composed of other phospholipids, such as dipalmitoylphosphatidylcholine (DPPC), phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, had no effect on hemolysis (Fig. 2).


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Fig. 1.   Lysenin-induced hemolysis. Erythrocytes (3 × 107 cells/ml) from various species (bullet , sheep; open circle , human; square , rat) were incubated with various concentrations of lysenin at 37 °C for 30 min, and the percentage of erythrocytes hemolyzed was determined by measuring the amount of hemoglobin released, as described under "Experimental Procedures."


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Fig. 2.   Inhibition of lysenin-induced hemolysis by sphingomyelin. Lysenin was preincubated with various concentrations of vesicles composed of 50 mol % each phospholipid and 50 mol % cholesterol, and the hemolytic activities of the resulting mixtures were measured. Phospholipids analyzed were as follows: bullet , sphingomyelin; open circle , DPPC; triangle , phosphatidylethanolamine; black-triangle, phosphatidic acid; square , phosphatidylserine; black-square, phosphatidylinositol.

The following observations (data not shown) also suggest that lysenin bound specifically to sphingomyelin on erythrocyte membranes and induced hemolysis. First, immunoblotting analysis showed that lysenin did not cross-react with cellular proteins of the erythrocyte membranes. Second, preincubation of lysenin with sheep erythrocyte membrane inhibited the lysenin-induced hemolysis, and treatment of the erythrocyte membrane with recombinant sphingomyelinase (Higeta Shoyu Co. Ltd., Japan) abolished its ability to inhibit the hemolysis. The high sensitivity of sheep erythrocytes to lysenin is attributable to the high sphingomyelin content in the sheep erythrocytes membranes; sphingomyelin is the predominant phospholipid (51% of total phospholipids) in sheep erythrocytes, whereas human and rat erythrocyte membranes contain much less sphingomyelin (25 and 13% of total phospholipids in human and rat erythrocyte membranes, respectively) (1).

Specific Binding of Lysenin to Sphingomyelin-- The specific binding of lysenin to sphingomyelin was confirmed by ELISA, TLC immunostaining, and liposome lysis assay. The ELISA showed lysenin bound to sphingomyelin and did not cross-react with other phospholipids (Fig. 3A). To define the specificity of lysenin further, we performed TLC immunostaining, since in the ELISA the sphingomyelin analogs, such as sphingosine 1-phosphate, were washed from the solid surfaces of the microtiter plates during the assay procedures. In the TLC immunostaining assay, the lipids coated onto high performance TLC plates were fixed by polyisobutylmethacrylate treatment, and almost all the lipids remained after the assay procedure (data not shown). As shown in Fig. 3B, lysenin bound specifically to sphingomyelin but not to the sphingolipid analogs, ceramide, sphingosine, sphingosine 1-phosphate, sphingosylphosphorylcholine, or galactosylceramide. When liposomes containing sphingomyelin were incubated with lysenin for 30 min at 37 °C, release of the entrapped marker, calcein, from the liposomes was observed (Fig. 4, A and B). The leakage of liposomes caused by lysenin was strictly dependent on the presence of sphingomyelin in the membranes, and no leakage was observed with liposomes containing sphingolipid analogs such as ceramide, sphingosine, sphingosine 1-phosphate, and galactosylceramide (Fig. 4B), suggesting that lysenin binds and perturbs the lipid bilayer structure only when sphingomyelin is present in the membranes. These results indicate clearly that lysenin recognized strictly the molecular structure of sphingomyelin and required phosphorylcholine, sphingosine, and fatty acid moieties for binding to sphingomyelin.


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Fig. 3.   Specific binding of lysenin to sphingomyelin. A, ELISA. The wells of the microtiter plate were coated with 500 pmol/well phospholipids and incubated with various concentrations of lysenin. Lysenin binding to the phospholipids was detected by sequential incubation with anti-lysenin antiserum, biotinylated anti-rabbit IgG, and peroxidase-conjugated streptavidin. Phospholipids analyzed were as follows: bullet , sphingomyelin; black-triangle, DPPC; triangle , distearoylphosphatidylcholine; black-square, phosphatidylethanolamine; square , phosphatidylserine; open circle , phosphatidylinositol; ×, phosphatidic acid. B, TLC immunostaining assay. 10 and 50 nmol of sphingomyelin and its analogs were spotted onto high performance TLC plates and incubated with 1 µg/ml lysenin. The bound lysenin was detected with anti-lysenin antiserum, biotinylated anti-rabbit IgG, and peroxidase-conjugated streptavidin. Lipids analyzed were as follows: sphingomyelin (1), ceramide (2), sphingosine (3), sphingosine 1-phosphate (4), sphingosylphosphorylcholine (5), and galactosylceramide (6).


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Fig. 4.   Lysis of liposomes containing sphingomyelin by lysenin. Liposomes (2 µM) containing calcein as a fluorescent marker were incubated with various concentrations of lysenin at 37 °C for 30 min, and the percentage of calcein released was determined by measuring the fluorescence of calcein, as described under "Experimental Procedures." A, liposomes were composed of phospholipids and cholesterol (molar ratio, 5:5). Phospholipids analyzed were as follows: bullet , sphingomyelin; open circle , DPPC; triangle , phosphatidylserine; black-square, phosphatidylinositol; square , phosphatidic acid. B, liposomes were composed of sphingolipids and DPPC and cholesterol (molar ratio, 1:4:5). Sphingolipids analyzed were as follows: bullet , sphingomyelin; open circle , ceramide; black-triangle, sphingosine; triangle , sphingosine 1-phosphate; black-square, galactosylceramide.

Hirota et al. (37) established a monoclonal antibody, VJ-41, which bound strongly to sphingomyelin but cross-reacted with phosphatidylcholine with saturated fatty acyl chains, such as DPPC. Sphingomyelin and DPPC, both of which are choline-containing phospholipids, exhibit similar thermotropic behavior; both are in a gel phase at 25 °C and liquid crystalline phase above 50 °C (1). It seems likely that this monoclonal antibody recognizes a particular topographical distribution of phosphorylcholine-containing lipids on membranes, including DPPC in a gel phase (37). In neither the ELISA (Fig. 3A) nor the liposome lysis assay (Fig. 4A) did lysenin cross-react with DPPC, again suggesting that lysenin recognizes a precise molecular structure of sphingomyelin. Recently Lange et al. (29) isolated a hemolysin, named as eiseniapore, from the coelomic fluid of E. fetida and showed that the protein caused lysis of liposomes containing sphingolipids (30). Lipid specificity of eiseniapore is quite different from that of lysenin, and eiseniapore showed a significant reactivity with both sphingomyelin and galactosylceramide but not with ceramide or galactosylsphingosine, suggesting that the ceramide structure as well as a hydrophilic moiety is essential for the interaction (30).

Molecular mechanism of the membrane damage caused by lysenin remains to be elucidated. Since some bacterial sphingomyelinases were shown to cause hemolysis (38), sphingomyelinase activity of lysenin was examined by using [14C]choline-labeled sphingomyelin as a substrate (39) or by measuring sphingomyelin contents in erythrocyte membranes after incubation with lysenin. No sphingomyelin degradation in either erythrocytes or sphingomyelin vesicles was observed after prolonged incubation with lysenin, suggesting that the lysenin-induced membrane damage did not result from the degradation of sphingomyelin. To examine whether lysenin picks up sphingomyelin from the membranes and serves as a soluble sphingomyelin carrier protein, lysenin was incubated with choline-methyl-14C-labeled sphingomyelin (NEN Life Science Products), followed by separation of free lysenin from the sphingomyelin vesicles by the spin column method developed by Chonn et al. (40). In this assay, no 14C-labeled sphingomyelin was detected in the free lysenin fraction, suggesting that lysenin does not pick up sphingomyelin from the membranes (data not shown). It is likely that lysenin caused the membrane damage by either perturbing the lipid bilayer structure or by forming aqueous pores in the membrane.

Effect of Cholesterol on the Lysenin-Sphingomyelin Interaction-- Sphingomyelin has been shown to associate preferentially with cholesterol in both artificial and biological membranes, and both lipids preferentially localized to plasma membrane of mammalian cells (41-48). To study the effect of cholesterol on the lysenin-sphingomyelin interaction, the hemolysis inhibition assay using sphingomyelin vesicles containing various amounts of cholesterol was performed. The concentration of sphingomyelin vesicles to cause 50% inhibition of hemolysis was reduced considerably when over 30 mol % of cholesterol was incorporated into the vesicles, whereas incorporation of less than 20 mol % had no significant effect, suggesting that the lysenin-sphingomyelin interaction was enhanced when over 30 mol % of cholesterol coexisted with sphingomyelin (Fig. 5).


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Fig. 5.   Effect of cholesterol on the lysenin-sphingomyelin interaction. Inhibition of lysenin-induced hemolysis by sphingomyelin vesicles containing various amounts of cholesterol was examined. Vesicles composed of sphingomyelin (black-square) or a mixture of sphingomyelin and cholesterol (molar ratios, 9:1, square ; 8:2, black-triangle; 7:3, triangle ; 6:4, open circle ; 5:5, bullet ) were preincubated with lysenin (10 ng/ml), and the hemolytic activities of the resulting mixtures were measured after incubation at 37 °C for 30 min.

A differential scanning calorimetry study of sphingomyelin- cholesterol vesicles (41) suggested that when less than 25 mol % cholesterol was incorporated into the vesicles, the two lipids exhibited phase separation, whereas they mixed when the sphingomyelin vesicles contained over 25 mol % cholesterol. The increased reactivity of lysenin with sphingomyelin when over 30 mol % cholesterol was present in the membranes may be due to a gel to liquid crystalline phase transition of the sphingomyelin-enriched domain that may have changed the topological distribution of sphingomyelin molecules, resulting in an increase in the number of sphingomyelin molecules accessible to lysenin. An alternative possibility is that lysenin recognized a sphingomyelin-cholesterol complex, more of which formed as the proportion of cholesterol increased.

Kinetic Analysis of the Lysenin-Sphingomyelin Interaction by Surface Plasmon Resonance Measurements-- To establish whether the enhancement of the lysenin-sphingomyelin interaction by cholesterol was due to a change in the topological distribution of sphingomyelin on the membranes or to a preferential interaction of lysenin with a sphingomyelin-cholesterol complex, we measured the lysenin-sphingomyelin binding parameters directly using BIAcoreTM system, an optical biosensor based on the principles of surface plasmon resonance (25). In this assay, the alkane thiol/gold surface of the sensor chip was coated with vesicles, and kinetic analysis of the lysenin-phospholipid membrane interaction in the presence of various concentrations of lysenin was performed. The resonance unit is an arbitrary unit used by the BIAcore system, and there is a linear relationship between the mass of protein bound to the sensor chip and the resonance unit observed (25). Lysenin binding to the phospholipid membrane was highly dependent on the presence of sphingomyelin in the membranes, and no significant binding was observed with the membrane composed of DPPC (Fig. 6A) nor with the membranes composed of other phospholipids such as phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol (data not shown). Although the amounts of lysenin bound to the membranes were increased in a dose-dependent manner of sphingomyelin in membranes (Fig. 6A), the dissociation constant of the lysenin-membrane interaction was not significantly affected by the sphingomyelin contents; KD values of lysenin with membranes containing 20, 50, and 80 mol % sphingomyelin in DPPC were 4.6, 6.6, and 3.2 nM, respectively. The lysenin binding to the membrane composed of sphingomyelin was increased 3-fold when cholesterol was incorporated into the membranes (Fig. 6B), indicating that the amount of lysenin bound to the membrane surfaces was increased by the presence of cholesterol. Results of the kinetic analysis of the cholesterol effect on lysenin-sphingomyelin interaction are summarized in Table I. Lysenin associated with membrane surfaces composed entirely of sphingomyelin with a kon value of 3.2 × 104 M-1 s-1 and dissociated extremely slowly with a koff value of 1.7 × 10-4 s-1, giving a low dissociation constant (KD = 5.3 nM). The incorporation of 50 mol % cholesterol into the sphingomyelin membranes did not alter the kinetics of the lysenin-membrane interaction significantly, only resulting in a slightly higher dissociation constant (KD = 8.6 nM). As the presence of cholesterol did not significantly affect the kinetic parameters of the lysenin-sphingomyelin interaction, it is likely that lysenin bound to sphingomyelin molecules, not to sphingomyelin-cholesterol complexes on the membrane surfaces, and that the enhanced association of lysenin with the sphingomyelin-cholesterol mixture was attributable to increased accessibility of sphingomyelin to lysenin due to the redistribution of sphingomyelin in the presence of cholesterol.


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Fig. 6.   Kinetic analysis of lysenin binding to membranes. Kinetic analysis of lysenin binding to immobilized phospholipid membranes was performed using BIAcoreTM system instrument (25). Small unilamellar vesicles composed of a combination of sphingomyelin (SM) and DPPC (A) or sphingomyelin and cholesterol (Chol.) at a molar ratio of 1:1 (B) were prepared in PBS. To immobilize the phospholipid layer on the sensor chip, the sensor chip was washed with 40 mM octyl glucoside, and vesicles containing 0.5 mM phospholipid were injected into the BIAcore system at a flow rate of 5 µl/min at 25 °C. Purified lysenin at the concentration of 1 µM was injected over the surface at a flow rate of 20 µl/min at 25 °C. The resonance unit is an arbitrary unit used by the BIAcore system, and there is a linear relationship between the mass of molecule bound to the sensor chip and the resonance unit observed (25).

                              
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Table I
Kinetic analysis of lysenin binding to sphingomyelin
Kinetic analysis of lysenin binding to immobilized phospholipid membranes was performed as described in the legend of Fig. 6 (25). The analyte binding assay was performed repeatedly after washing the surface with 30 mM NaOH using 50 µl at a flow rate of 20 µl/min. The binding kinetics were analyzed according to the manual of the software BIAevaluation 2.1. Each value represents the mean value of two different experiments.

Immunofluorescence Staining of Human Fibroblasts by Lysenin-- Niemann-Pick disease type A (NP-A) is an autosomal recessive disorder resulting from a deficiency of lysosomal acid sphingomyelinase activity, and NP-A fibroblasts have been shown to accumulate large amounts of sphingomyelin as well as cholesterol (49). Sphingomyelin accounts for 5-20% of the total cellular phospholipid content of most normal cells, but in cells of NP-A patients, the sphingomyelin level is elevated up to 50-fold, reaching about 70% of the total phospholipid fraction (50). Koval and Pagano (51) used a fluorescent analog of sphingomyelin and found that plasma membrane sphingomyelin was transported and accumulated in lysosomes in NP-A fibroblasts.

Cell-surface immunofluorescence staining of the NP-A and normal fibroblasts using lysenin showed that lysenin bound uniformly to plasma membrane of both normal and NP-A fibroblasts (Fig. 7A, a and c). When the cells were permeabilized by digitonin treatment, intense staining of cytosolic punctate organelle was observed with the NP-A fibroblasts (Fig. 7, B, a), and only weak staining of cell surface and cytosolic organelle was observed with normal fibroblasts (Fig. 7, B, d). The punctate organelles observed with the NP-A fibroblasts were co-stained with fluorescein-labeled dextran which was taken and stored in lysosomes (52) (Fig. 7, B, a and b), indicating that the punctate organelles were lysosomes. Lysenin binding was abolished when lysenin was preincubated with vesicles containing sphingomyelin (data not shown). The intense lysosomal staining of NP-A fibroblasts by lysenin is consistent with the previous observation that sphingomyelin accumulates predominantly in lysosomes of NP-A fibroblasts (51, 53), suggesting that lysenin can be used to detect sphingomyelin in animal cell membranes.


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Fig. 7.   Immunofluorescence staining of human fibroblasts by lysenin. A, fibroblasts from a patient with NP-A (a and b) or a normal subject (c and d) were fixed and stained by lysenin. Bound lysenin was detected by sequential incubation with anti-lysenin antiserum and tetramethylrhodamine isothiocyanate-conjugated anti-rabbit IgG. Phase contrast micrograph (b and d) and lysenin staining (a and c) of the same specimens are shown. (Bar = 20 µm.) B, fibroblasts from a patient with NP-A (a-c) or a normal subject (d-f), which had been cultured in the medium containing 5 mg/ml fluorescein-labeled dextran for 24 h, were fixed and permeabilized. Staining by lysenin was performed as described in A. Phase contrast micrograph (c and f) and lysenin staining (a and d) and dextran fluorescence (b and e) of the same specimens are shown.

The findings of this study provide the first concrete evidence that lysenin binds specifically to sphingomyelin. Lysenin will provide a useful tool for probing the molecular motion of sphingomyelin in cellular membranes and exploring the biological functions of sphingomyelin in studies on mutant cells with aberrant sphingomyelin metabolism or transport which will be selected by their altered sensitivity to lysenin.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 813-3823-2101 (ext. 5419); Fax: 813-3823-2130; E-mail: umeda{at}rinshoken.or.jp.

1 The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; NP-A, Niemann-Pick disease type A; DPPC, dipalmitoylphosphatidylcholine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBS, Tris-buffered saline.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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