(Received for publication, December 2, 1996, and in revised form, June 10, 1997)
From the Lytic activity in the coelomic fluid of earthworm
(Eisenia fetida fetida) has been ascribed to eiseniapore, a
hemolytic protein of 38 kDa. Since receptors for eiseniapore on target
cell membranes are not known, we used lipid vesicles of various
composition to determine whether specific lipids may serve as
receptors. Lytic activity of eiseniapore was probed by the relief of
fluorescence dequenching from the fluorophore
8-aminonaphthalene-1,3,6-trisulfonic acid originally incorporated
into the vesicle lumen as a complex with
p-xylene-bis-pyridinium bromide. Hemolysin binds to and
disturbs the lipid bilayer only when distinct sphingolipids consisting of a hydrophilic head group as phosphorylcholine or galactosyl as well
as the ceramide backbone, e.g. sphingomyelin, are present. Cholesterol enhances eiseniapore lytic activity toward
sphingomyelin-containing vesicles probably due to interaction with
sphingomyelin. Leakage of vesicles was most efficient when the lipid
composition resembled that of the outer leaflet of human erythrocytes.
Presumably, an oligomeric protein pore formed by six monomers is
responsible for leakage of sphingomyelin-containing vesicles. The
secondary structure of eiseniapore did not change upon binding to lipid membranes. The lytic activity of eiseniapore was completely abolished after its denaturation or after preincubation with polyclonal antibodies. Our results suggest that the presence of specific sphingolipids is sufficient to mediate lytic activity of eiseniapore. This action contributes to our understanding of earthworm immune responses.
Coelomic fluid of the earthworm Eisenia fetida ssp.
(Oligochaeta, Lumbricidae) containing more than 40 proteins exhibits
several biological effects as follows: cytolytic, proteolytic,
hemolytic, hemagglutinating, tumorstatic, mitogenic, and bacteriostatic
activities (1-10). Recently, efforts have been directed toward
identifying the molecular nature and regulation of lytic activity.
Based upon partial purification of coelomic fluid, cytolytic and
hemolytic activities have been associated with a protein of an apparent molecular mass of about 42 kDa (11). We have isolated recently a
hemolysin from the coelomic fluid of Eisenia fetida fetida, referred to as eiseniapore, for the first time (12). The molecular mass
of this thiol-activated hemolysin was 38 kDa, and it exerted a strong
lytic activity against erythrocytes.
At least two different mechanisms preventing homologous lysis by
eiseniapore have to be considered. First, an eiseniapore-regulating factor (ERF) purified recently by us inhibits the eiseniapore hemolytic
properties (12). Eiseniapore-regulating factor shares immunological
properties with vitronectin, an inhibitor of complement and perforin
lytic activities (13, 14). Indeed, vitronectin and eiseniapore
interacted reversibly to suppress hemolytic activity, and
eiseniapore-regulating factor acted as a potent inhibitor of
complement-induced hemolysis. Identification of an
eiseniapore-regulating factor and its properties confirms the
hypothesis of Canicatti (15), which suggested that vitronectin and
related structures act as possible protectors of autolysis in
invertebrates. Second, another strategy that may prevent self-killing
considers the absence of membrane-associated receptor(s), which are
required for binding of eiseniapore to self-membranes. However, the
receptor for eiseniapore and a mechanism of any subsequent interaction
with the lipid bilayer are not identified. Glycoproteins of the cell
membrane are suggested as receptors since various acetylated and
methylated carbohydrates were effective inhibitors of hemolytic
activity of coelom fluid (1). Moreover, preincubation of semi-purified
hemolysin with sphingomyelin, a typical component of mammalian plasma
membranes, strongly suppressed hemolysis of mammalian erythrocytes (4). These results prompted the hypothesis of a specific involvement of
sphingomyelin in the hemolysin-mediated lysis of target cells (15). As
a corollary, it is not known whether this lipid will act as a receptor
for eiseniapore.
Here we present a comprehensive analysis concerning the interaction of
eiseniapore from E. fetida fetida with unilamellar liposomes
of various lipid composition. We discovered for the first time that
distinct sphingolipids are an absolute requirement for both binding of
eiseniapore to lipid membranes and their subsequent leakage. In the
presence of these lipids we observed the formation of oligomeric
pore-like structures of eiseniapore on liposomal membranes and rapid
leakage of vesicles. While cholesterol alone did not mediate binding of
nor leakage by eiseniapore, complexes of sphingomyelin and cholesterol
enhanced its lytic activity. Our data suggest that eiseniapore
hemolytic activity is optimized toward a lipid composition typically
for the outer leaflet of the plasma membrane of mammalian erythrocytes.
Most importantly, our results imply that membrane-(glyco)proteins, as
receptors for eiseniapore, are not required as long as specific
sphingolipids are present.
Adult E. fetida fetida (58), synonym E. fetida typica (59)
(Annelida, Oligochaeta, Lumbricidae), were collected from 15 localities
throughout Northern Germany. Earthworms were washed, cultured, and
covered with wet filter paper for 48 h at 6 °C. Coelomic fluid
was obtained by puncturing the coelomic cavity with a glass
micropipette. The suspension pooled from 40 earthworms was centrifuged
(13.000 × g, 17 min) and the supernatant used immediately for purification of eiseniapore.
Preparative
PAGE1 ensures high yield
purification of biologically active molecules. Since we found that SDS
destroyed the lytic activity of
eiseniapore,2 it was omitted.
We found that the pI of eiseniapore was below 8.5, and the lytic
activity of eiseniapore was not impaired up to a pH of 9.5. Thus, we
used the Ornstein-Davis system (Tris/chloride/glycine) (16, 17) as
follows: upper and lower electrode buffer (25 mM Tris base,
192 mM glycine), elution buffer (25 mM Tris
base, 192 mM glycine, 2 mM reduced glutathione,
1.5 mM thioglycolic acid), and sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 0.025% bromphenol
blue, 2 mM reduced glutathione, 2 mM
thioglycolic acid, 4 mM phenylmethylsulfonyl fluoride).
Addition of reduced glutathione and thioglycolic acid was required to
protect the biological activity of the protein. The monomer
concentration for maximum resolution was determined empirically with
native analytical gels. The system used for preparative electrophoresis was composed of a running gel (38-ml monomer volume of 7% acrylamide, 0.18% bisacrylamide) and a stacking gel (5-ml monomer volume of 4%
acrylamide, 0.106% bisacrylamide). Preparative electrophoresis (model
491 Prep Cell, Bio-Rad) was conducted at 15 watts for 13.5 h with
a flow rate of 1 ml/min. Fractions of the elution buffer were collected
at 4 °C and tested for hemolytic activity of eiseniapore using a
suspension of 2% sheep erythrocytes in a microtiter plate assay.
Hemolytic fractions were monitored by using 5-10% SDS-PAGE (see
below), and purified hemolysin was used for dialysis and lyophilization.
SDS-PAGE (nonreducing
condition) was performed with 5-10% acrylamide gels in a vertical gel
apparatus (Protean 2 xi Vertical Electrophoresis Cells; Bio-Rad).
Samples were prepared by mixing equal volumes of sample and buffer
(62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5%
Rabbits were immunized
subdermally into the backs with 20 µg of isolated eiseniapore in
incomplete Freund's adjuvant. An additional immunization was made 2 weeks later. Sera were purified by using a protein A-Sepharose column
(Bio-Rad) with a final concentration of antibodies of 0.5 mg/ml. To
probe neutralization, samples (80 µl) of eiseniapore (5 µg/ml) were
diluted with 40 µl of antibody solution (100 µg/ml) and incubated
for 20 min at 25 °C.
CD spectra were recorded on a JASCO
J-720 spectropolarimeter interfaced with an external water bath to
maintain temperature control and connected to a PC for data recording
and processing. Fused silica cells of 1 mm path length were used.
Spectra were recorded from 195 to 250 nm using a scan speed of 10 nm/min. The spectra correspond to the average of three scans. For data
processing, the base line was subtracted, and spectra were smoothed
using the software package supplied with the spectropolarimeter. The protein concentration was 3 µM in 10 mM
sodium phosphate, pH 7.2.
Fluorescence was
measured at 25 °C with an Aminco Bowman Series 2 Luminescence
Spectrometer (Rochester, NY) using 10-mm quartz cuvettes. Protein
fluorescence intensity was measured at wavelengths of Vesicle membranes in the absence
(control) and presence of eiseniapore were fixed with 1%
glutaraldehyde at room temperature. Fixation was stopped after 10 min
by addition of 1 M Tris-HCl, pH 7.4. Specimens were
visualized with 1% di-sodium phosphotungstate, pH 7.0, applying a
double-carbon film technique. As support, 400-mesh copper grids covered
with a holey carbon film were used. Micrographs were taken with an EM
400T electron microscope (Philips, Eindhoven, The Netherlands) at 80 kV
with a magnification of 60,000 and 80,000 ×.
Lipids (Sigma,
Deisenhofen, Germany) of the desired composition (total lipid
concentration 750 µmol/liter) were dissolved in 1 ml of
chloroform/methanol (1:1) and subsequently dried under nitrogen at room
temperature. Lipids were hydrated by the addition of phosphate-buffered
saline (5.8 mM
NaH2PO4/Na2HPO4, 150 mM NaCl, pH 7.4). If not stated otherwise small unilamellar
vesicles (SUV) have been used. SUV were prepared by sonification of the
lipid suspension on ice with a Branson sonifier (Danbury, CT) until the
suspension became opalescent, usually for 8 min (18). Large unilamellar
vesicles (LUV) of different composition were prepared after six
freeze-thaw cycles by extrusion (extruder from Lipex Biomembranes
Inc., Vancouver, Canada) through two stacked polycarbonate membranes
(Nucleopore, CA) with 0.1-µm pore size. ANTS
(8-aminonaphthalene-1,3,6-trisulfonic acid, di-sodium salt) · DPX
(p-xylene-bis-pyridinium bromide) (Molecular Probes, OR)
-filled vesicles and ghosts, respectively, were used to measure
eiseniapore-induced leakage of luminal aqueous contents. The
incorporation of the water-soluble complex ANTS·DPX into vesicles and
the leakage assay were performed following the method described in
Ellens et al. (19). Vesicles contained 12.5 mM
ANTS, 45 mM DPX, 20 mM NaCl, and 10 mM Tris-HCl, pH 7.4. Vesicles were separated from
non-encapsulated ANTS·DPX complexes by chromatography on Sephadex
G-75 with Tes buffer (100 mM NaCl, 2 mM Tes, 2 mM L-histidine, and 1 mM EDTA, pH
7.4). The same buffer but with 0.1 mM EDTA only was used
for the leakage assay. For preparing resealed ghosts we followed the
approach of Schwoch and Passow (20) with the modifications of Pomorski
et al. (21). Briefly, lysis of 2 ml of ice-cold suspension
of washed erythrocytes (90% packed) in 1.2 mM acetic acid,
4 mM MgSO4, 1.2 mM ATP, 1 mM CaCl2 (pH~ 3) was performed at 4 °C.
After 5 min erythrocytes were spun down, and the pellet was resuspended
in buffer (12.5 mM ANTS, 45 mM DPX, 50 mM Tris-HCl) and pH 7.2 was adjusted by addition of
appropriate amounts of 0.1 M NaOH. Resealing of ghosts was performed by incubation at 37 °C for 45 min. Ghosts were separated from non-incorporated fluorescence marker by gel filtration (Sephadex G-75 column) using 50 mM Tris-HCl, 85 mM NaCl,
and 10 mM CaCl2, pH 7.0, as elution buffer. To
study the influence of lysolipids on the interaction of eiseniapore
with ghost membranes, ghosts (total lipid concentration 750 µmol/liter) were preincubated with L- Leakage measurements were performed
using a Shimadzu RF5001PC spectrofluorometer (Duisburg, Germany) at
various temperatures with the wavelengths set to To characterize membrane fluidity of
liposomes, we recorded the EPR membrane spectrum of the spin-labeled
fatty acid I(12, 3) (5-doxyl-stearic acid; Sigma, Deisenhofen,
Germany). The paramagnetic NO moiety is localized in the hydrophobic
membrane phase close to the head group region of lipids. The
concentration of I (12, 3) was 1 mol % of endogenous lipids. EPR
spectra were measured by a Bruker ECS 106 (Bruker, Karlsruhe, Germany)
or by a Radiscope RS100 (Magnettech GmbH, Berlin, Germany) both
equipped with a temperature controller. From the spectra the order
parameter was measured which has been shown to characterize membrane
fluidity (22); an increase in fluidity causes a decrease of this
parameter. The order parameter S was calculated according to
Equation 1.
Universität Greifswald, Zoologisches
Institut und Museum, Bachstrasse 11/12, D-17489
Greifswald, Federal Republic of Germany, the ¶ Laboratory of
Comparative Immunology, Department of Neurobiology, UCLA Medical
Center, University of California,
Los Angeles, California 90095-1763, the
§ Humboldt-Universität zu Berlin,
Max-Delbrück-Centrum für Molekulare Medizin,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Earthworms and Harvesting of Coelomic Fluid
-mercaptoethanol) and subsequent boiling for 5 min. Gels were run at
250 V.
ex = 280 nm and
em = 330 nm for excitation and emission, respectively. Background intensity measured in the absence of protein was subtracted.
-lysopalmitoylphosphatidylcholine (C16:0, Sigma,
Deisenhofen, Germany) for 7 min at 25 °C before gel filtration.
ex = 355 nm and
em = 530 nm, respectively. Dilution of the
ANTS·DPX complexes caused by their release from the vesicle or ghost
lumen leads to complex dissociation and, by that, to the relief of
fluorescence quenching of ANTS. Thus, leakage is accompanied by an
increase of ANTS fluorescence intensity. To elucidate the percentage of
leakage, at the end of each experiment Triton X-100 (Sigma,
Deisenhofen, Germany) was added (final concentration 0.1 vol %) to
allow infinite dilution and dissociation of the ANTS·DPX
complexes.
with A
(Eq. 1)
and A|
the outer and inner hyperfine splitting, respectively, obtained from
spectra. Azz (33 G) and Axx (6.95 G) are the respective hyperfine tensors, and a0
(15.10 G) is the isotropic hyperfine splitting of the crystal (23).
We measured leakage
of small unilamellar vesicles (SUV) in the presence of eiseniapore
using the ANTS · DPX assay. We observed no eiseniapore-mediated
destabilization and, thus, leakage of liposomes consisted only of egg
phosphatidylcholine (egg-PC) (Fig. 1,
curve a, pH 7.4, 25 °C, molar lipid to protein ratio
(L/P) = 50:1 (mol/mol)). Even at an L/P as low as 10:1, eiseniapore caused no leakage of egg-PC liposomes. Likewise, variation in temperature, 10-37 °C, and pH, 4.8-8.1, did not affect the
stability of egg-PC membranes in the presence of eiseniapore.
Furthermore, no eiseniapore-mediated release of ANTS · DPX was
observed upon incorporation of phosphatidylserine (PS) and/or
phosphatidylethanolamine (PE) up to 50 mol % into egg-PC liposomes
(data not shown). When replacing egg-PC for
dipalmitoylphosphatidylcholine also no lysis of liposomes was observed
neither below nor above the phase transition of this phospholipid (data
not shown) indicating that the phase state and/or membrane fluidity of
the lipid bilayer did not determine the lytic activity of
eiseniapore.
A profound leakage of liposomes was observed, however, in the presence
of sphingomyelin (SM) (Fig. 1). At an egg-PC/SM ratio of 3:1 the extent
of leakage after 7 min was approximately 10% (Fig. 1, curve
b, 25 °C, pH 7.4, L/P = 50:1). An increase in ANTS · DPX leakage occurred when the lipid ratio was changed from 3:1 to 1:3 (Fig.
1, curves c and d). Leakage of
sphingomyelin-containing vesicles was also found at much higher L/P
ratios (Fig. 2, curve b, L/P
250:1, Fig. 4). Destabilization of egg-PC/SM liposomes by eiseniapore
was efficient at all L/P ratios investigated in the presence of
cholesterol (e.g. L/P = 250:1, pH 7.4, 25 °C, Fig.
2). Both the extent (see also Fig. 4) and the kinetics of leakage were
enhanced in liposomes composed of egg-PC/Chol/SM, but there was no
leakage in the case of egg-PC/Chol (Fig. 2) nor PS/egg-PC/Chol
liposomes. Since it is known that cholesterol exerts a profound
influence on membrane fluidity, the latter observations support the
preliminary conclusion that the membrane fluidity does not resemble a
determinant of eiseniapore interaction with membranes.
The extent of lysis of sphingomyelin-containing liposomes by
eiseniapore depends upon the molar lipid to protein ratio. The leakage
process became accelerated, and the final extent of lysis was enhanced
upon increasing the protein concentration relative to lipids as shown
for PS/egg-PC/Chol/SM (1:1:2:4) vesicles in Fig.
3A (see also Fig.
4). For kinetics, we measured the
half-time of vesicle lysis corresponding to 50% of the extent of final
leakage. Dependence of half-time on the inverse of protein
concentration can be fitted to a first approximation by a linear
function (Fig. 3B, curve a). The extent of leakage is a
function of molar lipid to protein ratio demonstrated for egg-PC/SM
liposomes of various compositions (Fig. 4).
Recently, we discovered that eiseniapore caused hemolysis of human erythrocytes (12) which prompted the preparation of liposomes resembling in their lipid composition the erythrocyte outer leaflet. The main lipid components in the exoplasmic human erythrocyte membrane leaflet are cholesterol, phosphatidylcholine, and sphingomyelin (24, 25). Moreover, phosphatidylethanolamine and gangliosides (GA) are present, whereas phosphatidylserine is almost exclusively oriented to the cytoplasmic leaflet (25). To mimic the exoplasmic leaflet of the erythrocyte membrane liposomes were made of egg-PC/Chol/SM/PE/GA (12:17:10:3:1). Indeed, those vesicles allowed high eiseniapore activity (Fig. 4). For example, L/P as low as 260:1 facilitated a significant leakage of about 40% after 7 min incubation at 25 °C (pH 7.4). Even at 15 °C, the extent was about 35% (data not shown). Remarkably, a sphingolipid as sphingomyelin was required for eiseniapore to exhibit lytic activity. Neither gangliosides nor the phospholipids phosphatidylethanolamine and phosphatidylserine could replace sphingomyelin. The presence of phosphatidylserine in sphingomyelin-containing liposomes, however, stimulated eiseniapore-induced leakage (Fig. 4).
To elucidate whether high curvature of SUV is essential for eiseniapore-mediated leakage, we also probed large unilamellar vesicles (LUV). Using LUV composed of PS/egg-PC/Chol/SM (1:1:2:4), we observed a slight reduction in the extent of leakage when compared with SUV (25 °C, pH 7.4; Fig. 4, inset). This indicates that curvature of lipid membranes is not an essential determinant for destabilization of lipid membranes by eiseniapore.
To prove that leakage of lipid vesicles is specifically related to eiseniapore, we preincubated the protein with polyclonal antibodies (see "Materials and Methods"). After adding of ANTS · DPX-containing liposomes (egg-PC/Chol/SM, 1:1:2) no leakage was observed. Denaturation of eiseniapore by preincubation at 56 °C in each instance caused a complete loss of its lytic activity suggesting that the native conformation of eiseniapore is essential for its activity.
Eiseniapore-mediated release of ANTS · DPX from
sphingomyelin-containing liposomes depended upon pH and temperature. As
shown for PS/egg-PC/Chol/SM (1:1:2:4), liposomal leakage (Fig.
5A, 25 °C) at neutral pH
reached a maximum. At acidic or alkaline pH, pH 4.8 and pH 8.1, respectively, extent of lysis as well as kinetics declined. While the
eiseniapore-induced release of ANTS/DPX from vesicles was slow with a
low final extent at 10 °C, leakage became faster and enhanced after
increasing the temperature (Fig. 5B, measured for 25 and
37 °C, respectively).
By thin layer chromatography (data not shown) we verified that no destruction of lipids occurred during the time course of the experiments. Thus, we have no indication for a hydrolase activity of eiseniapore.
Eiseniapore-induced Leakage of Resealed Erythrocyte GhostsSince (i) eiseniapore caused hemolysis of human
erythrocytes (12) and (ii) liposomes comparable in their lipid
composition to the outer leaflet of erythrocytes are sensitive to
eiseniapore (see above), we measured leakage of resealed human
erythrocyte ghosts using the ANTS · DPX assay. As can be deduced from
the kinetics and the extent of leakage (Fig.
6A, 25 °C, pH 7.4),
eiseniapore-triggered release of fluorophore from ghosts was faster and
more efficient in comparison to liposomes even for a lipid composition
corresponding to the outer leaflet of human erythrocytes (see also Fig.
3B). Even at the low L/P of 16,000:1, we found a significant
leakage (about 20% after 7 min). At a ratio of 100:1, leakage was
high; after 1.5 min of the addition of eiseniapore, leakage attained approximately 80% close to its plateau (90%). To a first
approximation the half-time of the kinetics (see above) depends also
linearly upon the inverse of the eiseniapore concentration (Fig.
3B).
To determine whether the composition of the lipid phase was also crucial for the interaction with eiseniapore in the case of ghosts, we preincubated ANTS · DPX filled ghosts with 0.5 mol % lysophosphatidylcholine (C16:0). By employing radioactively labeled lysophosphatidylcholine, we showed the rapid insertion of those lipids into ghosts membrane (data not shown).3 The presence of 0.5 mol % lysophosphatidylcholine in ghost membranes caused a significant reduction of eiseniapore-mediated leakage (Fig. 6B). Similar to liposomes, we observed no leakage of ghosts upon denaturation of eiseniapore by heat pretreatment (data not shown).
Binding of Eiseniapore to LiposomesTo ascertain whether
binding of eiseniapore to sphingomyelin-containing liposomes was
reversible, we measured the ANTS · DPX leakage of liposomes after
preincubation of eiseniapore with unlabeled vesicles. When eiseniapore
was preincubated with sphingomyelin-free vesicles (egg-PC, egg-PC/Chol
(1:1), or egg-PC/PS (1:1)) for 15 min at 25 °C, the subsequent
addition of labeled egg-PC/Chol/SM (1:1:2) caused a release of
ANTS · DPX that was similar in extent and kinetics to that of
controls (preincubation in the absence of liposomes) (Fig.
7). However, after preincubation of
eiseniapore with egg-PC/Chol/SM (1:1:2) liposomes subsequently added
ANTS · DPX-labeled vesicles of the same lipid composition caused no
lysis (Fig. 7). This suggests an irreversible binding of eiseniapore to
sphingomyelin-containing liposomes in agreement with results obtained
by electron microscopy (see below).
As shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of
eiseniapore, irreversible binding to liposomes may be explained by the
formation of high molecular weight complexes of eiseniapore. When the
protein was incubated in the presence of sphingomyelin-containing vesicles, we observed bands of a molecular mass of about 228 kDa which
is about six times higher than that of a monomer (38 kDa) (Fig.
8). This suggests the formation of
oligomers consisting of six monomers. Bands of higher molecular weight
complexes were not detected when eiseniapore was incubated with lipid
vesicles lacking sphingomyelin.
Electron Microscopy
The interaction of eiseniapore with
vesicles was further investigated by electron microscopy of
glutaraldehyde-fixed negatively stained samples. In the presence of
eiseniapore we observed circular structures in the lipid bilayer of
liposomes provided sphingomyelin was incorporated into the membrane
(Fig. 9). We suggest that these structures represent oligomeric complexes of eiseniapore. They were not
observed in the absence of sphingomyelin. The outer and inner diameter
of the pore-like structures are approximately 10 and 2-3 nm. In side
views, eiseniapore complexes appear as square-like structures with a
height of about 10 nm, which project from the edge of the vesicle
membrane. Thus, eiseniapore complexes seem to form a cylindrical
structure with a central hole oriented perpendicular to the surface of
the vesicle membrane (Fig. 9, b-d).
Molecular Components of Sphingomyelin Essential for Leakage
We elucidated which structural features of sphingomyelin are important for interaction with lipid membranes and investigated whether complexes of cholesterol and sphingomyelin are responsible for enhanced extent of leakage when cholesterol is incorporated into sphingomyelin liposomes. We measured the extent of ANTS · DPX leakage in the presence of different sphingolipids at various temperatures and molar lipid to protein ratios. Almost the same extent was measured when sphingomyelin was replaced by galactosylceramide in egg-PC liposomes, but the single chain galactosylsphingosine (psychosine) was ineffective in mediating leakage (Table I, 37 °C, L/P, 250:1). Therefore, the ceramide structure with two chains seems to be essential. However, the presence of ceramide alone was not sufficient because no lytic activity of eiseniapore was detected when sphingomyelin was replaced by the double-chain N-palmitoyl-D-sphingosine. Thus, the ceramide structure as well as a hydrophilic moiety seemed to be essential membrane components for eiseniapore to mediate leakage of liposomes.
|
No further increase of fluorophore release was observed when cholesterol was added to phosphatidylcholine liposomes containing galactosylceramide in contrast to the significant enhancement after incorporation of sphingomyelin (Table I). The data suggest that interaction between cholesterol and sphingomyelin, e.g. by formation of complexes (see "Discussion"), may enhance eiseniapore lytic activity. Results similar to that shown in Table I were obtained at lower temperature (25 °C) (data not shown).
For the various liposomes, we measured the order parameter of EPR membrane spectra of the spin-labeled fatty acid I(12, 3) at 37 °C (Table I; spectra not shown). This parameter provides a measure of membrane fluidity. The order parameter did not correlate with leakage. Thus, in agreement with the above results, we can rule out that membrane fluidity is an essential determinant of eiseniapore lytic activity.
Characterization of Eiseniapore in the Presence of LiposomesA fluorescence spectrum typical for tryptophan residues
was measured when eiseniapore was excited at 280 nm (Fig.
10A). Presumably, tryptophan
residues are buried within the protein in a rather hydrophobic region.
This is supported by the fluorescence maximum at 333 nm which is
characteristic for an apolar environment. Addition of the aqueous
quencher potassium iodide (20 mM final concentration) caused a reduction of approximately 8% in fluorescence intensity. No
influence on fluorescence intensity nor on wavelength of its maximum
was observed in the presence of liposomes which did not contain
sphingomyelin. However, a significant decrease in
eiseniapore-associated fluorescence was observed when egg-PC/Chol/SM
liposomes were added (Fig. 10B). The wavelength of
fluorescence maximum was only slightly shifted to higher values
(max = 335 nm). Upon binding of eiseniapore to those
liposomes, we found an enhanced accessibility of potassium iodide to
tryptophan residues. The fluorescence intensity was decreased by
approximately 30% in the presence of 20 mM KI.
The CD spectrum of eiseniapore was measured in the presence or absence
of liposomes consisting either of egg-PC/Chol/SM or only of PC. No
alteration of the CD spectrum of eiseniapore was observed after adding
lipid vesicles even when sphingomyelin and cholesterol were
incorporated (data not shown). The secondary structure of eiseniapore
consists of about 37% -sheet, 28%
-helix, 17%
-turn, and
18% random coil as estimated by the computer program CONTIN (26).
We have shown that a purified hemolysin (eiseniapore) from the earthworm E. fetida fetida (58) is able to perturb the bilayer structure of liposomal as well as human erythrocyte membranes causing release of internal aqueous contents from each of them. Its lytic activity does not require any other component from the coelom fluid, and it is suppressed by corresponding polyclonal antibodies. However, this activity is unambiguously associated with its native structure; it was completely lost after denaturation of eiseniapore by heat pretreatment. An essential prerequisite for eiseniapore-mediated leakage of lipid membranes was the presence of distinct sphingolipids as sphingomyelin or galactosylceramide. Sphingolipids as sphingomyelin are essential and, presumably, sufficient to cause leakage of lipid membranes by eiseniapore. In the absence of sphingomyelin, no release of egg-phosphatidylcholine liposomal content was detected even in the presence of phosphatidylserine, phosphatidylethanolamine, and cholesterol, respectively. A rapid leakage of liposomes was observed only after incorporation of sphingomyelin which was maximum at neutral pH. Although more detailed investigations are warranted, we elucidated the following molecular features of sphingolipids as imperative for eiseniapore-mediated leakage: (i) a hydrophilic head group as phosphorylcholine or galactosyl and (ii) the double chain backbone of sphingolipids, the ceramide.
A significant enhancement of eiseniapore-mediated leakage of egg-PC/SM liposomes was observed when cholesterol was present. Cholesterol alone did not support lytic activity because (i) in the absence of sphingomyelin no liposomal release was observed, and (ii) leakage was not enhanced when sphingomyelin was replaced by galactocerebroside. These results suggest that the interaction, perhaps the formation of complexes, between cholesterol and sphingomyelin is responsible for increased leakage after incorporating cholesterol into egg-PC/SM liposomes. Recently sphingomyelin has been shown to form complexes with cholesterol (27, 28), whereas galactoceramide does not (29, 30, see also Ref. 31). Remarkably, the cholesterol interaction of sphingomyelins and galactoceramides may depend significantly on the structure of the acyl chains (32).
We observed the highest extent of leakage when the lipid composition of liposomes simulated that of the outer leaflet of human erythrocyte ghosts. The lytic activity was even higher for resealed human erythrocyte ghosts. At present we do not know which components of the erythrocyte membrane are responsible for this additional stimulating effect on eiseniapore-mediated lysis. We precluded that the lytic activity of eiseniapore was related to a specific phase state of the bilayer. Furthermore, electrostatic interactions do not seem to play an essential role since phosphatidylserine alone could not render egg-PC liposomes susceptible for eiseniapore and was not required for leakage of SM liposomes. We cannot preclude the possibility that specific protein receptors may exist which enable eiseniapore to develop its lytic activity. However, as long as distinct sphingolipids are present those proteins are not required. Although no conclusive data are available, so far no indication for the existence of protein receptors for eiseniapore has been given. The present study points rather to sphingolipids as the relevant target for eiseniapore. Indeed, we found that eiseniapore-triggered hemolysis of erythrocytes of different mammalian species was closely related to the sphingomyelin content of the erythrocyte membrane.4
Our results do not only emphasize the essential role of sphingolipids for leakage but also strongly support that these lipids are necessary for binding. Preincubation of eiseniapore in the presence of liposomes of various lipid composition but without sphingomyelin did not affect the subsequent eiseniapore-mediated leakage of egg-PC/Chol/SM liposomes. In contrast, no leakage of labeled egg-PC/Chol/SM liposomes was found when eiseniapore was preincubated with unlabeled liposomes of the same composition. Thus, we surmise that binding of eiseniapore to liposomes containing sphingolipids as sphingomyelin is essentially irreversible. We have no evidence for substantial binding of eiseniapore to lipid membranes lacking specific sphingolipids.
Presumably, membrane-bound eiseniapore forms an oligomeric pore-like structure provided sphingolipids like sphingomyelin are present. These complexes seem to consist of six monomers as deduced from PAGE and are rather stable because they were degraded into monomers only after 30 min of boiling in the presence of reducing agents (data not shown). By electron microscopy, the complexes were visualized as regular structures on sphingomyelin-containing liposomes with an inner and outer diameter of about 2-3 and 10 nm. We observed similar complexes of eiseniapore on sheep erythrocyte membranes4 and suggest that these complexes are pores through which leakage may proceed. This is supported by our recent results on the osmotic protection of eiseniapore-triggered hemolysis of sheep erythrocytes (12). Protection from colloid osmotic lysis by sugars or polymers of different molecular weights suggests an effective pore diameter of about 3 nm in erythrocytes. The formation of multimeric complexes responsible for leakage is also supported by the half-time of leakage depending (to a first approximation) linearly on the inverse eiseniapore concentration suggesting that the protein acts at some stage, at least, as a dimer. As already pointed out (33, 34) the formation of protein oligomers often occurs via protein dimerization. However, further studies are warranted to determine whether eiseniapore-mediated leakage involves cooperative interaction of several monomers.
The secondary structure of eiseniapore contains predominantly
-structures. Such a high portion of
-sheets is not unusual for
proteins interacting with membranes in an oligomeric form. For example,
poly-C9 of the complement system (35), staphylococcal
-toxin (36),
aerolysin, a cytolytic bacterial exotoxin produced by Aeromonas
hydrophila (37), and the
-toxin of Clostridium perfringens (perfringolysin O) (38) exhibit a high content of
-sheet structure. We have no indication for a rearrangement in secondary structure of eiseniapore upon binding to
sphingomyelin-containing lipid membranes as deduced from the far UV-CD
spectrum. However, we cannot rule out that changes of the secondary
structure of various parts of eiseniapore may compensate each other
and, thus, become invisible in the spectrum. In contrast, we found
moderate alterations of tryptophan fluorescence which may indicate some reorientation of tertiary and/or quaternary structure. A rearrangement of tertiary structure of membrane active proteins without a significant change in secondary structure has been observed for the pore-forming bacterial toxins colicin A and aerolysin (39-41).
Our results on the interaction of eiseniapore with sphingomyelin have important implications for understanding the immune response of earthworms. First, we propose that a self-killing activity of eiseniapore may be prevented by an altered molecular structure of sphingolipids. Of course, an efficient mechanism would consider the absence of those lipids from respective target membranes of annelids. Although no comprehensive lipid analysis of various earthworm tissues is available, apparently sphingolipids represent only a minor component of annelid membranes (42-44) and, more specifically, of Eisenia fetida (45). For instance, Okamura et al. (46) found that the earthworm nervous system does not contain sphingomyelin. Second, earthworm parasites, i.e. ciliates, cestodes, and nematodes, do not survive in the coelomic fluid. Remarkably, sphingolipids are typical lipids for those parasites (47-51). Third, our analysis suggests a strategy that could answer whether eiseniapore receptors different from sphingolipids exist. For example, cells such as bacteria, sensitive to whole coelomic fluid (3, 52) but lacking sphingolipids, are interesting biological systems for those studies. Finally, we emphasize that the general conclusion on distinct sphingolipids as receptors for hemolysins of annelids and other invertebrates is awaiting the confirmation by future studies using hemolysins from other species (see also Refs. 53-57).
We thank Prof. Dr. Gregor Damaschun and Dr. Dietrich Zirwer (Max-Delbrück-Center, Berlin-Buch) for assistance in CD measurements and Rudolf Erlemann for helpful discussion.