From the Institute of Medical Microbiology and
Hygiene, University of Mainz, Obere Zahlbacher Strasse 67, D55101
Mainz, Germany, and § Department of Chemistry and
Biochemistry, Queens College of The City University of New York,
Flushing, New York 11367-1597
Received for publication, January 10, 2001, and in revised form, February 1, 2001
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ABSTRACT |
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Vibrio cholerae cytolysin
(VCC) forms oligomeric pores in lipid bilayers containing cholesterol.
Membrane permeabilization is inefficient if the sterol is embedded
within bilayers prepared from phosphatidylcholine only but is greatly
enhanced if the target membrane also contains ceramide. Although the
enhancement of VCC action is stereospecific with respect to
cholesterol, we show here that no such specificity applies to the two
stereocenters in ceramide; all four stereoisomers of ceramide enhanced
VCC activity in cholesterol-containing bilayers. A wide variety of
ceramide analogs were as effective as
D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of
lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast,
the activity of staphylococcal To bacterial pore-forming cytolysins, cholesterol is a logical
choice as a target molecule, because it confers specificity for animal
as opposed to bacterial cell membranes. The specificity for cholesterol
is shared between Vibrio cholerae cytolysin
(VCC)1 (1) and streptolysin O
(SLO) (2). Otherwise, these toxins are not related, and the
oligomeric pores they form are very different in size and morphology
(1, 3). Although with SLO the sterol is already required in the initial
event of membrane binding of the monomeric toxin (4), it only comes
into play at the stage of oligomerization in the case of VCC (5, 6).
When the sterol is incorporated into phosphatidylcholine (PC) bilayers
at physiologically realistic concentrations (i.e.
up to 40% by mol), these membranes do not become significantly
sensitive to VCC. However, it was previously found that membrane
susceptibility toward the cytolysin was greatly enhanced by inclusion
of ceramide; free ceramide and monohexosyl ceramides proved similarly
effective (7). A combined specificity for cholesterol and sphingolipids
has previously been shown for the fusion protein of Semliki Forest
virus. In that instance, the interaction with ceramide proved to be
highly stereoselective (8-10). Accordingly, we have examined the
structural properties of the ceramide molecule responsible for the
sensitization of membranes to VCC. To our surprise, no dependence on
stereospecific features of ceramide could be detected. Membrane
sensitization was readily achieved with a variety of synthetic
ceramides and even with 1,2-diacyl-sn-glycerol, which is not
closely related in structure to ceramide. However, with both sphingo-
and glycerolipids, the presence of a phosphocholine headgroup led to a
decrease in membrane susceptibility toward VCC. In glycerolipids other
than PC and diacylglycerol, headgroups smaller in size than
phosphocholine were associated with higher membrane susceptibility to
the cytolysin. Among lipid species sharing the same headgroup, a
complementary trend was generally apparent in which acyl chains having
a large cross-section were associated with higher VCC activity,
although there were significant exceptions to this correlation.
Remarkably, despite its lack of a structural homology with VCC, SLO was
affected in its activity toward cholesterol-containing bilayers in a
very similar way by the incorporation of sphingo- and glycerolipids into the bilayer. We propose that the effects of sphingo- and glycerolipids on membrane susceptibility to VCC and SLO do not arise
because of any direct, specific effect upon the toxins but instead are
mediated by their ability to enhance the interaction of cholesterol
with the toxins.
Purification of VCC, SLO, and Lipids--
1-(16-Indolyl-palmitoyl)-2-oleoyl-sn-glycero-3-phosphocholine
and
1-(16-bromo-palmitoyl)-2-oleoyl-sn-glycero-3-phosphocholine were kindly supplied by Dr. John Silvius, McGill University. The synthesis of the four stereoisomers of n-C8:0-ceramide (see
Fig. 1) were described previously (13) and characterized as the
N-biphenylcarboxamido derivatives of sphingosine by
high pressure liquid chromatography (14).
D-erythro-Triple bond-C16-Cer (see Fig. 1) was
synthesized as described previously (15).
D-erythro-Aryl-C4-Cer (see Fig. 1) was
synthesized as described recently (16). The other ceramide analogs
shown in Fig. 1 were prepared by using the synthetic sequences outlined
below. EYPC, EYPG, bovine brain (2S,3R)-4E-1,3-Dihydroxy-2-N-capryloylamino-4-octadecene
(D-erythro-C8-Cer)--
Into a round-bottomed flask were
combined 25 mg (0.08 mmol) of
D-erythro-sphingosine, 24 mg (0.09 mmol) of
p-nitrophenyl caprylate, and 5 ml of anhydrous
tetrahydrofuran. The reaction mixture was stirred at room temperature
for 24 h, at which time TLC analysis indicated the consumption of
sphingosine (silica gel-coated aluminum plates, eluted with ethyl
acetate (Rf = 0.73) and visualized with 10%
sulfuric acid in methanol). The reaction mixture was concentrated on a
rotary evaporator. The residue was dissolved in 1 ml of a 25% solution
of ethyl acetate in hexanes, which was loaded onto a silica gel column
(10 × 100 mm). The column was eluted with 100 ml of 25% ethyl
acetate in hexanes and then with 200 ml of ethyl acetate. The fractions
containing the product were collected and concentrated to afford 33 mg
(97%) of the product as a colorless solid. The film was lyophilized
from 5 ml of anhydrous benzene to afford a colorless powder.
1H NMR (400 MHz, CDCl3) (2S,3R,4S,5S)-2-N-Capryloylamino-4,5-cyclopropyl-1,3-dihydroxyoctadecane
(D-erythro-Cyclopropyl-C8-Cer) and Its Mono-O-methyl
Analog--
Into a 25-ml round-bottomed flask 1.3 ml of a 15%
(by weight) solution of diethylzinc (1.16 mmol) in n-hexane
was added to a stirring solution of 110 µl (1.31 mmol) of
diiodomethane in 5 ml of dichloromethane at room temperature under
argon. After the reaction mixture was stirred for 15 min, 33 mg (77 µmol) of N-C8:0-D-erythro-ceramide
was added as a solution in 1.0 ml of dichloromethane. After 1 h
the reaction was quenched by the addition of 5 ml of 0.5 M
H2SO4. The aqueous phase was separated and
extracted with three 25-ml portions of ethyl acetate. The combined
organic extracts were dried over MgSO4 and concentrated
under reduced pressure. The residue was chromatographed on silica (1:1
hexanes:ethyl acetate) to give 20 mg (59%) of the product
(Rf = 0.53) and 14 mg of its
mono-O-methyl analog (Rf = 0.74),
each as colorless solids (total yield, 99%). Each of the two solids was lyophilized from 5 ml of benzene to afford colorless amorphous powders. D-erythro-Cyclopropyl-C8-Cer:
1H NMR (400 MHz, CDCl3) (2S,3R)-4E-1,3-Dihydroxy-2-n-(heptanesulfonylamino)-4-octadecene
(D-erythro-Sulfonamido-Cer)--
Into a 5-ml
round-bottomed flask was charged 300 mg (1.5 mmol) of sodium
heptanesulfonate and 2.0 ml of thionyl chloride. A single drop of
anhydrous N,N-dimethylformamide was added, and the solution was heated to reflux under argon for 5 h. The
unreacted thionyl chloride was removed by distillation under argon. The residue was dissolved in 5 ml of anhydrous diethyl ether, and the
solution was filtered through sea sand. The resulting solution was
concentrated under reduced pressure to provide
n-heptanesulfonyl chloride as a clear yellow oil. A 28-mg
portion of the sulfonyl chloride was dissolved in 1.0 ml of
dichloromethane and was added to a 5.0-ml dichloromethane solution
containing 25 mg (0.08 mmol) of
D-erythro-sphingosine and 10 µl of pyridine.
The solution was stirred for 12 h at room temperature under argon.
The reaction mixture was concentrated under reduced pressure, and the
residue was chromatographed on silica (1:1, hexanes:ethyl acetate,
Rf = 0.89) to provide 15 mg (41%) of the
product as an off-white solid. The latter was lyophilized from 5 ml of
benzene to provide the product as an off-white powder.
1H NMR (400 MHz, CDCl3) Preparation of LUV--
The lipids were dissolved in chloroform
and mixed at the molar ratios indicated under "Results" (total
amount, 5 mg) and dried with nitrogen in a round-bottom flask to form a
thin film. The lipids were resuspended in 10 mM HEPES/100
mM NaCl/50 mM calcein (pH 7.5) by warming, if
necessary, and bath sonication. The suspensions were frozen and thawed
and then repeatedly extruded through polycarbonate membranes
(Nuclepore; pore size 100 nm) using a 10-ml thermobarrel extruder
(Lipex, Vancouver, Canada). The non-entrapped calcein was removed by
gel filtration on Sephadex G50 in 10 mM HEPES/10 mM NaCl (pH 7.5). The lipid concentration of the final
liposome suspension was quantified by using a commercial enzymatic
assay of cholesterol (Roche Molecular Biochemicals).
Calcein Release Assay--
From a solution of the cytolysin in
question (200 µg/ml in HEPES/NaCl with 0.1% bovine serum albumin),
2-fold serial dilutions were prepared. To each dilution, an equal
volume of the respective preparation of calcein liposomes (total lipid
content, 0.2 mg/ml) was added. Following incubation for 10 min at
37 °C, the samples were diluted into 30 volumes of HEPES/NaCl, and
the calcein fluorescence was assayed in a SPEX Fluorimax fluorometer
(excitation wavelength, 488 nm; emission, 520 nm). The extent of
calcein release was calculated from the relative increase of
fluorescence intensity over a sample of untreated liposomes, whereby
the fluorescence intensity corresponding to 100% permeabilization was
assayed on a sample lysed with sodium deoxycholate (final
concentration, 6 mM). The amounts of toxin required for 25 or 50% release of calcein (RD25 or RD50,
respectively) were determined from plots of permeabilization
versus toxin dosage by linear interpolation from the two
adjacent data points.
Analysis of Lipid Phase Separation by Fluorescence
Quenching--
Liposomes were prepared from C12- and
C20-ceramide, respectively (molar content, 30%),
cholesterol (35%), EYPC (32%), and EYPG (2%), and the fluorescently
labeled phospholipid (16-indolyl-palmitoyl)- oleoyl-PC (1%; total
amount of lipids, 200 nmol). Parallel samples contained the quenching
lipid (16-Br-palmitoyl)-oleoyl-PC instead of EYPC. The LUV were assayed
for fluorescence (wavelengths were as follows: excitation, 280 nm;
emission, 320 nm) in HEPES/NaCl buffer. An aliquot of each sample was
dissolved with 10 volumes of methanol; the fluorescence intensity of
this sample was used to normalize the intensity of the undissociated
sample. The decrease in fluorescence intensity by the quenching lipid
with C12- and C20-ceramide, respectively, was
then determined from the normalized values. The assay was performed
likewise with dipalmitoyl-PE and dilauroyl-PE in place of
C20- and C12-ceramide, respectively.
Sphingomyelinase Treatment and Hemolysis of Sheep
Erythrocytes--
Sheep erythrocytes were made up to 20% (by volume)
with 10 mM HEPES, 140 mM NaCl, 2 mM
CaCl2, 1 mM MgCl2, pH 7.0. They
were incubated for 40 min at 30 °C with 2,5, or 10 milliunits/ml sphingomyelinase (Bacillus cereus; EC
3.1.4.12; Sigma). They were then washed three times with the former
buffer (without CaCl2 and MgCl2) by centrifugation. After resuspension to 2%, VCC was added to 1.5 µg/ml. Hemolysis was followed over time by cell turbidity
(A600).
Lack of Effect of Chiral and Structural Modifications in Ceramide
on VCC Activity in LUV--
In a previous study, it was found that
addition of ceramide to PC bilayers containing cholesterol (35% by
mol) enhanced VCC-induced permeabilization (7). To determine the
structural features of the ceramide molecule responsible for this
sensitizing action, we tested a variety of synthetic ceramide
derivatives. Fig. 1 shows the structures
of the synthetic ceramide analogs we used. The ceramides were added to
20 mol % to give a uniform background mixture of lipids comprising
cholesterol (35 mol %), EYPG (2 mol %), and EYPC (making up the
remainder to 100 mol %). From these lipid mixtures liposomes loaded
with the aqueous fluorescent marker calcein were prepared as described
previously (7), and the release of calcein was determined as a function
of VCC concentration. Fig. 2A
shows that replacement of the
D-erythro-ceramide (which represents
the physiological configuration) by the
L-erythro, D-threo, or
L-threo isomer does not cause a major change in
liposome membrane susceptibility, indicating that there is no
stereospecific interaction between ceramide and VCC.
The dosages of VCC required to induce the release of calcein to 50%
(RD50) or 25% (RD25) were calculated to
provide a concise estimate of the effect of various lipids on membrane
susceptibility to VCC. Fig. 2B gives the RD50
values obtained with further molecular modifications in ceramide
structure. It is evident that changing the stereochemistry of the C4=C5
double bond of the sphingoid chain from trans to
cis or its replacement by a triple bond, a cyclopropyl
group, or even a benzene ring does not significantly change the
activity of VCC. This finding is in clear contrast to the known role of
the double bond in the support of viral fusion (10, 17), as well as in
activator protein-1 activation by sphingosylphosphocholine and
sphingosine-1-phosphate (18) and in the induction of apoptosis in
leukemic cells (14, 19, 20). Similarly, reversing the positions of the
amido and secondary hydroxy functionalities or replacement of the amide
bond with a sulfonamide group do not have major effects on the activity of VCC either. Fig. 2B also shows that even
1,2-dioleoyl-sn-glycerol brings about a similarly strong
sensitization as do the ceramides. Thus, if ceramide and
dioleoylglycerol share a common mode of action, the stereochemical
features of either molecule are surely not important.
Role of the Phospholipid Headgroup and Fatty Acyl Chains on VCC
Activity--
It was previously found that sphingomyelin was less
effective than ceramide in sensitizing membranes to VCC, suggesting an inhibitory role of the phosphocholine headgroup (7). The finding that
diacylglycerol is more effective than PC indicates that the phosphocholine moiety is inhibitory not only in sphingolipids but also
in glycerolipids.
In mixed lipid bilayers with cholesterol, the headgroups of
phospholipids are thought to shield the steroid ring system of cholesterol from water (21). Cholesterol shielded from water might then
also be shielded from VCC, which may account for the inhibitory effect
of the phosphocholine headgroup. In comparisons of cholesterol
solubility in PC and PE bilayers, it was proposed that PC, because of
its larger headgroup, would make a more efficient "umbrella" for
the sterol than PE (22). The effect of phospholipid headgroup size on
the activity of VCC was examined with the dioleoyl species of the
following glycerophospholipids: phosphatidic acid, phosphatidylserine,
phosphatidylglycerol, PE, N-methyl-PE, and N,N'-dimethyl-PE. A plot of the RD25
values of VCC observed with these lipids (at 20 mol %) against their
theoretical headgroup volumes (Fig.
3A) shows a clear relationship
between phospholipid headgroup volume and cytolysin dosage
(i.e. an inverse relationship of headgroup volume
and membrane susceptibility), which is consistent with the umbrella
concept.
Another way to increase the "umbrella-shielded space" available to
the sterol would be to decrease the cross-sectional area of the fatty
acyl chains. The variation of membrane susceptibility to VCC on
changing the acyl chain cross-section was evaluated with various
synthetic PEs. The results are shown in Fig. 3B. Clearly,
dilauroyl-PE and dimyristoyl-PE have a lower sensitizing effect than
dioleoyl-PE, which in turn is exceeded by diphytanoyl-PE, a synthetic
lipid with multiple methyl branches and hence particularly bulky acyl
chains (phytanic acid:3,7,11,15-tetramethylhexadecanoic acid). This
direct relation of acyl chain cross-section and membrane susceptibility
to VCC is also consistent with the umbrella model. However,
dimyristoyl-PE (as well as dipalmitoyl-PE; not shown) also have longer
acyl chains than dilauroyl-PE, yet are slightly less effective than the latter.
The effect of acyl chain length was also studied with
ceramides (Fig. 3C). A direct relationship between acyl
chain length and membrane susceptibility was maintained from
N-acetyl-sphingosine to the N-lauroyl derivative.
However, when the chain length was increased further (at 20 mol % ceramide), membrane susceptibility to VCC decreased. A regular
relationship between the fatty acyl chain length and membrane
susceptibility can only be expected if homogeneous mixing is assumed to
take place for all of the lipid species present in the membrane
bilayer. However, both glycerolipids (23, 24) and ceramides (25, 26)
may undergo lateral phase separation. Lipid molecules bearing long,
highly saturated acyl chains are particularly prone to undergo
segregation within the bilayer, which might explain the decrease of
membrane susceptibility with ceramide acyl chain length increasing
beyond 12 carbons. Liposomes containing 30 mol % of
C12-ceramide or C20-ceramide were examined
using a fluorescence-quenching assay of lipid phase separation. These
liposomes also included two labeled PC species, the fluorescent
compound (16-indolyl-palmitoyl)-oleoyl-PC, and the
fluorescence-quenching species (16-bromo-palmitoyl)-oleoyl-PC. In
similar experiments involving cerebrosides, this pair of probes was
found to display enhanced fluorescence quenching on phase separation,
which was ascribed to their cosegregation into the cerebroside-depleted
phase (27). The fluorescence intensity was indeed more strongly
quenched with C20-ceramide, the relative intensity being
70% with respect to C12-ceramide. This suggests that
C20-ceramide does not mix ideally in the bilayer and
segregates more readily than C12-ceramide. Segregation of
C20-ceramide into ceramide-rich regions would lower the
effective concentration of this lipid that is available for
interactions with other membrane components and may therefore account
for the lower degree of membrane sensitization effected by
C20-ceramide. Similarly, dipalmitoyl-PE appeared to undergo
phase separation more readily than dilauroyl-PE (the relative
fluorescence being 0.75 in this case), which may likewise account for
the lower membrane susceptibility observed with dipalmitoyl-PE.
Lateral segregation of a lipid sparingly soluble in the bulk phase of
the membrane is less likely to occur when its concentration is
decreased. Fig. 3C shows that, indeed, C16- and
C20-ceramides are slightly more active than
C12-ceramide at 5 mol %. The finding of significant
membrane susceptibility to VCC with such low levels of ceramide is
quite remarkable in its own right; similar observations were made with
the most effective of the glycerolipids tested, dioleylglycerol (data
not shown). The data support the notion that the molecular shape of the
lipid influences the activity of VCC on the target membrane. Lipids
that have a conical molecular shape, i.e. those
whose hydrophobic moiety occupies a larger cross-section than does the
polar headgroup, enhance membrane susceptibility to VCC. That this
effect may be significant not only with synthetic but also with natural
membranes is exemplified in Fig. 3D. With sheep
erythrocytes, enzymatic conversion of sphingomyelin to ceramide strongly enhances VCC-mediated hemolysis.
Effect of Phospho- and Glycerolipid Molecular Shape on the
Activities of Streptolysin O and Staphylococcal Effect of Enhanced Cholesterol Content and Augmentation of Membrane
Susceptibility to Toxins by Cholesteryl Acetate--
In the absence of
any ceramides or glycerolipids, both VCC and SLO are still highly
active when the concentration of membrane cholesterol is raised from 35 to 55 mol % (data not shown). A very similar effect can be obtained by
supplementation of 35 mol % cholesterol with 20 mol % cholesteryl
acetate. However, cholesteryl acetate is a very poor substitute of
cholesterol as a specific ligand for SLO or VCC (data not shown),
because the 3- The present results do not confirm our previous proposal that VCC
displays a dual specificity for cholesterol and ceramide (7). In marked
contrast to the fusion protein of Semliki Forest virus, which truly
displays such dual specificity (8, 29), VCC is not significantly
affected even by major structural alterations in the ceramide molecule.
Nevertheless, ceramides and some glycerolipids strongly augment the
activity of VCC, up to several hundred times, in the presence of modest
membrane concentrations of cholesterol.
Lee et al. (30) studied the impact of various headgroups on
bilayer stability in binary mixtures of PE and the respective phospholipid in question; the mixed membranes were monitored for hexagonal-II phase transition. It is instructive to compare their results with the effects of the same lipids on cytolysin activity (Fig.
5). With all cytolysins examined, there
is a correlation between the ability of lipids to promote the
hexagonal-II phase and cytolysin activity. Because -hemolysin, which does not share with
the other toxins the requirement for cholesterol, was far less affected
by the presence of lipids with a conical shape. The collective data
indicate that sphingolipids and glycerolipids do not interact with the
cytolysins specifically. Instead, lipids that have a conical molecular
shape appear to effect a change in the energetic state of membrane
cholesterol that in turn augments the interaction of the sterol
with the cholesterol-specific cytolysins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Hemolysin--
VCC and
-hemolysin were purified from bacterial culture supernatants as
described previously (5, 11). SLO was expressed recombinantly in
Escherichia coli as a maltose-binding protein fusion protein
and purified from bacterial cell lysates (12).
-galactosylceramide, bovine brain
non-hydroxy and
-hydroxy fatty acyl ceramides, and cholesterol
acetate were purchased from Sigma. All other lipids were obtained from
Avanti Polar Lipids (Alabaster, AL).
6.35 (NH, d,
J = 7.3 Hz), 5.77 (1H, dt, J = 14.5, 6.7 Hz), 4.30 (1H, m), 3.91 (2H, m), 3.68 (1H, dd, J = 10.7, 2.7 Hz), 2.22 (2H, t, J = 7.4 Hz), 2.05 (2H, dt,
J = 14.0, 6.9 Hz), 1.63 (2H, m), 1.26 (30H, m), 0.88 (6H, t, J = 6.5 Hz); 13C NMR (100 MHz,
CDCl3)
174.1, 134.2, 128.8, 74.4, 62.4, 54.6, 36.8, 32.3, 31.9, 31.7, 29.7 (3), 29.68, 29.67, 29.5, 29.4, 29.3, 29.28, 29.26, 29.18, 29.06, 25.8, 22.7, 14.1.
6.43 (NH, d,
J = 7.7 Hz), 4.13 (1H, dd, J = 11.4, 3.2 Hz), 3.98 (1H, m), 3.78 (1H, dd, J = 11.3, 2.8 Hz), 3.17 (1H, dd, J = 8.1, 3.1 Hz), 2.22 (2H, dd,
J = 7.8, 7.5 Hz), 1.64 (4H, m), 1.26 (30H, m), 0.88 (6H, m), 0.75 (2H, m), 0.52 (1H, m), 0.37 (1H, m); 13C NMR
(100 MHz, CDCl3)
173.7, 78.1, 62.7, 54.0, 38.7, 36.8, 33.6, 31.9, 31.7, 29.73 (4), 29.69, 29.57, 29.48, 29.40, 29.28, 29.0, 28.9, 25.7, 22.7, 22.2, 16.7, 14.1, 14.0, 10.4. Mono-O-methyl-D-erythro-cyclopropyl-C8-Cer:
1H NMR (400 MHz, CDCl3)
6.27 (NH, d,
J = 8.0 Hz), 4.06 (1H, m), 3.90 (1H, dd,
J = 9.7, 3.4 Hz), 3.59 (1H, dd, J = 9.7, 2.7 Hz), 3.33 (3H, s) 3.08 (1H, m), 2.21 (2H, m), 1.62 (4H, m), 1.26 (30H, m), 0.86 (6H, m), 0.65 (2H, m), 0.54 (1H, m), 0.34 (1H, m); 13C NMR (100 MHz, CDCl3)
173.1, 77.0, 73.0, 59.3, 52.3, 36.9, 33.6, 31.9, 31.7, 29.71 (4), 29.68, 29.55, 29.51, 29.4 (2), 29.2, 29.0, 25.7, 22.7, 22.6, 22.1, 16.2, 14.14, 14.08, 9.9.
5.82 (1H,
dt, J = 15.4, 6.8 Hz), 5.50 (1H, dd,
J = 15.4, 6.1 Hz), 5.17 (NH, d, J = 8.5 Hz), 4.66, (OH, br s), 3.91 (1H, dd, J = 11.5, 3.9 Hz),
3.72 (1H, m), 3.43 (1H, m), 3.08 (2H, m), 2.64 (OH, br s), 2.62 (OH, br
s), 2.06 (2H, dt, J = 14.1, 7.0 Hz), 1.83 (2H, m), 1.38 (4H, m), 1.26 (26H, m) 0.88 (6H, m); 13C NMR (100 MHz,
CDCl3)
134.8, 128.0, 74.8, 62.6, 58.4, 53.8, 32.3, 31.9, 31.5, 29.71, 29.70, 29.68, 29.63, 29.5, 29.38, 29.37, 29.26, 29.0, 28.8, 28.3, 23.7, 22.7, 22.5, 14.1, 14.0.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structures of
D-erythro-ceramide and of the synthetic
ceramide analogs used in this study.
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Fig. 2.
Effect of structural modifications of
ceramide on the activity of VCC. A, susceptibility of
liposomes (LUV) containing D-erythro-ceramide,
or its stereoisomers, to permeabilization by VCC (calcein release
assay). Liposomes consisted of cholesterol (35%), ceramides as
indicated (20%), EYPG (2%), and EYPC (to 100 mol %) and were loaded
with calcein (50 mM). They were incubated with various
amounts of VCC, and permeabilization was quantified by means of the
increase in fluorescence of the calcein released. B,
susceptibility of LUV containing synthetic ceramide derivatives or
dioleoylglycerol. LUV membrane composition was as above, as was the
assay method. From the release curves, RD50 was determined
by interpolation. A low RD50 value corresponds to high
membrane susceptibility.
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Fig. 3.
Role of lipid headgroup and acyl chains in
the activity of VCC. A, relationship of glycerolipid
headgroup size and VCC activity. Liposomes (LUV) consisted of
cholesterol (35%), various glycerolipids (as their dioleoyl species)
as indicated (20%), EYPG (2%), and EYPC (remainder to 100 mol %).
The glycerolipid species were as follows: 1,
dioleoylglycerol; 2, phosphatidic acid; 3, PE;
4, PG; 5, N-methyl-PE; 6, PS;
7, N,N-dimethyl-PE; 8, PC.
Membrane permeabilization was assayed as above (see legend to Fig. 2),
except that the RD25 was determined. B,
VCC-mediated permeabilization of liposomes containing PE with various
acyl chains at 20 mol %, respectively. DphPE,
diphytanoyl-PE; DLPE, dilauryl-PE; DOPE,
dioleoyl-PE; DMPE, dimyristoyl-PE. C, ceramide
acyl chain length and VCC activity. Liposomes were prepared with
cholesterol/EYPG/EYPC as before but with synthetic ceramides carrying
saturated acyl chains varying in length from C2 through
C20. The molar fraction of ceramide in the membrane was
either 20 or 5%, as indicated. D, effect of
sphingomyelinase pretreatment on hemolysis of sheep erythrocytes by
VCC. Sheep erythrocytes (2% by volume) were pretreated for 40 min at
30 °C with various amounts of sphingomyelinase and then exposed to
VCC (1.5 µg/ml). Hemolysis was assayed by the decrease in turbidity
(A600).
-Hemolysin--
We
propose that the putative mode of action of ceramides and glycerolipids
is an indirect one; they change the state of membrane cholesterol and
only thereby alter the activity of VCC, which depends on cholesterol.
To test this hypothesis, we examined the effects of ceramides and of
glycerolipids on another cholesterol-specific toxin. SLO belongs to a
class of cholesterol-specific toxins (4) that are not at all
structurally similar to VCC. Fig.
4A shows that, indeed, SLO is
affected by the presence of ceramides in the target membranes in a
fashion that closely resembles VCC. This is also found with neutral
glycerolipids, whereas with acidic glycerolipids the relationship is
somewhat different (Fig. 4B). Finally, Fig. 4C
compares the respective efficacy of the two toxins with all the
liposomes that were tested with both VCC and SLO. As the lipids we used
vary considerably in structure (comprising ceramides, neutral and
charged glycerolipids, and even cholesteryl acetate; see below), the
correlation coefficient of 0.73 is significant. For comparison, we
examined a cholesterol-independent toxin, staphylococcal
-hemolysin.
It is evident that in this case the correlation with VCC is much
weaker, which suggests that lipid molecular shape is not as important a
determinant of the activity of staphylococcal
-hemolysin.
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Fig. 4.
Susceptibility of liposome membranes to VCC:
correlation with SLO and with staphylococcal
-hemolysin. A, SLO and VCC.
Liposomes (LUV) consisted of cholesterol (35%), EYPG (2%), various
synthetic ceramides (20%), and EYPC (to 100 mol %). The ceramide
species are as follows: 1, C2; 2,
C6; 3, C20; 4,
C16; 5, C8; 6,
C12. The correlation coefficient (R2) was
calculated on the logarithms of the respective RD50 values.
B, RD25 of SLO and of VCC on membranes
containing various dioleoyl glycerolipids (at 20 mol %) in bilayers
from EYPC, EYPG, and cholesterol (cf. A).
Filled circles indicate neutral lipids, and open
circles represent acidic lipids. The glycerolipid species
are as follows: 1, dioleoylglycerol; 2, PE;
3, N-methyl-PE; 4,
N,N-dimethyl-PE; 5, PC; 6,
phosphatidic acid, 7, PS; 8, PG. The solid
line (R2 = 0.67) fits both neutral and acidic
glycerolipids, and the broken line (R2 = 0.89)
covers the neutral lipids. C, RD25 of SLO, VCC,
and
-hemolysin on membranes containing ceramides, glycerolipids, or
cholesteryl acetate at 20 mol % (residual membrane components as
before).
-hydroxy group is an important determinant of
stereospecificity with both VCC (1) and SLO (2, 28). This suggests that
the sensitizing effect of supplementation of the cholesterol content
does not involve stereospecific interaction between the toxins and the additional sterol.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hemolysin is not
strictly dependent on cholesterol, there may be a
non-cholesterol-specific component in the effect of bilayer stability
on pore-forming toxins. Such a component would be in agreement with
recent findings on aerolysin, which, like
-hemolysin, does not
strictly require membrane cholesterol, although those results are not
directly comparable in quantitative terms (31). However, the
correlation between the phase transition temperature and toxin activity
is much steeper and much more consistent with the two
cholesterol-specific toxins than with
-hemolysin (Fig. 5). This
finding strongly suggests that the sterol participate in the observed
modulation of toxin activity.
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Fig. 5.
Effects of
1,2-dioleoyl-sn-3-glycerolipids with various
headgroups on lipid bilayers: correlation of hexagonal-II phase
transition temperature with cytolysin activity. The phase
transition temperatures, as determined with mixed bilayers of
dioleoyl-PE and the glycerolipid in question, were taken from Ref. 30.
The glycerolipid species are as follows: 1,
dioleoylglycerol; 2, phosphatidic acid; 3, PE;
4, PG; 5, PS; 6, PC. Correlation
coefficients were calculated from the logarithms of the
RD25 values, which were determined with the glycerolipids
contained at 20% within the standard cholesterol/EYPG/EYPC lipid
mixture (see Fig. 3A).
How, then, is cholesterol linked to bilayer stability? Like all of the activating lipids, cholesterol is a cone-shaped lipid, i.e. it claims space in the hydrophobic layer but does not provide for adequate headgroup coverage, which it must borrow from adjacent lipids. Thus, cholesterol molecules within a bilayer compete with all other cone-shaped molecules for headgroup coverage, no matter whether these are glycerolipids, sphingolipids, different sterols, or other cholesterol molecules. The latter case occurs in membranes with high cholesterol content. Shortage of headgroup coverage will expose the sterol to an energetically unfavorable state; as a consequence, the free energy of cholesterol is likely to rise steeply with its membrane concentration (21). Eventually, cholesterol will dissociate from the bilayer and precipitate, which takes place more readily with PE than with PC bilayers (22), in accordance with the notion that PC makes a better umbrella for the sterol (21). The binding sites of cytolysins may offer another opportunity for cholesterol molecules to seek shelter from the unfavorable contact with water. To the extent that the sterol is closely embraced by the cytolysin, cholesterol will in effect leave the bilayer and hence get rid of the associated chemical potential. Thus, the tighter the interaction between the sterol and the protein, the more sensitive the protein becomes to the cholesterol free energy, and, in turn, to the thermodynamic stability of the bilayer.
The proposed model rationalizes the effects of a wide variety of lipids and of variations in the cholesterol concentration (32) under a single common principle. It may also accommodate the previous finding that, at constant lipid composition, VCC is more active in small unilamellar vesicles than in LUV (7), because membrane curvature also preferentially strains the headgroup layer. Finally and importantly, there is no reason why the proposed mechanism of indirect activation by cone-shaped lipids should be restricted to bacterial toxins. Any other membrane-associated protein possessing a specific binding site for cholesterol may also be affected by alterations of the cholesterol chemical potential in a similar manner. In this context, it is noteworthy that ceramide and diacylglycerol are particularly effective activators of VCC and SLO. These lipids are both generated in the cytoplasmic membrane by phospholipases and act as messenger molecules by binding to specific acceptor proteins. However, the major fraction of these lipids will remain dispersed in the membrane, where they accordingly should increase the free energy of cholesterol. Because various membrane proteins, e.g. receptors for oxytocin or acetylcholine, are endowed with specific binding sites for cholesterol (33-35), this raises the intriguing possibility that cholesterol may function as a second messenger to diacylglycerol and/or ceramide within the cytoplasmic membrane.
Nevertheless, it must be stressed that the proposed hypothesis needs to
be tested by direct measurements of the chemical potential of
cholesterol in mixed lipid bilayers. So far, however, it appears that
there is no reliable method for this test. Because spontaneous partitioning of cholesterol between vesicles of different composition is slow (36-38), the sterol may not reach equilibrium before other lipid species start to mix by fusion or redistribution. Although methyl- and hydroxypropyl--cyclodextrin catalyze depletion of cholesterol from membranes or its insertion into them (39, 40), cyclodextrins may interact directly with membrane lipids and may thus
alter the energetic state of the membrane-associated sterol.
In conclusion, the present study shows that the activity of
cholesterol-specific bacterial cytolysins on mixed bilayers may be
strongly enhanced by lipids other than cholesterol. It provides evidence that these accessory lipids do not act upon the toxins specifically but instead modulate the interaction of the toxins with
cholesterol. The proposed mode of coupling between cholesterol and
cone-shaped lipids within mixed bilayers may be of broad interest, because it could similarly apply to the regulation of cellular membrane
proteins endowed with cholesterol binding sites,
e.g. hormone or neurotransmitter receptors.
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ACKNOWLEDGEMENT |
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We thank Dr. John Silvius (McGill University) for samples of labeled phospholipids.
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FOOTNOTES |
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* This study was supported by the Deutsche Forschungsgemeinschaft (SFB 490) and by the National Institutes of Health (Grant HL-16660).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: Dept. of Medical Biochemistry and Genetics, Texas A & M University, 440 Reynolds Medical Bldg., College Station, TX 77843-1114. Tel.: 979-847-8935; Fax: 979-847-9481; E-mail: mpalmer@medicine.tamu.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M100241200
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ABBREVIATIONS |
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The abbreviations used are: VCC, Vibrio cholerae cytolysin; SLO, streptolysin O; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; C2-ceramide, D-erythro-N-acetylsphingosine; C12-ceramide, D-erythro-N-dodecylsphingosine; C16-ceramide, D-erythro-N-hexadecylsphingosine; C20-ceramide, D-erythro-N-arachidylsphingosine; EYPC, egg yolk PC; EYPG, egg yolk PG; LUV, large unilamellar vesicles; RD25 (RD50), cytolysin dosage required for release of 25% (50%) of calcein from vesicles.
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