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INTRODUCTION |
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
,
-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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS AND DISCUSSION |
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 ( , sheep;
, human; , 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: ,
sphingomyelin; , DPPC; , phosphatidylethanolamine; ,
phosphatidic acid; , phosphatidylserine; ,
phosphatidylinositol.
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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: , sphingomyelin; , DPPC; ,
distearoylphosphatidylcholine; , phosphatidylethanolamine; ,
phosphatidylserine; , 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: , sphingomyelin; , DPPC;
, phosphatidylserine; , phosphatidylinositol; , phosphatidic
acid. B, liposomes were composed of sphingolipids and DPPC
and cholesterol (molar ratio, 1:4:5). Sphingolipids analyzed were as
follows: , sphingomyelin; , ceramide; , sphingosine; ,
sphingosine 1-phosphate; , galactosylceramide.
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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 ( ) or a
mixture of sphingomyelin and cholesterol (molar ratios, 9:1, ; 8:2,
; 7:3, ; 6:4, ; 5:5, ) 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.
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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.
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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.
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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.