From the Institute of Medical Microbiology, University of Mainz, Augustusplatz, D55101 Germany
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ABSTRACT |
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Vibrio cholerae cytolysin
permeabilizes animal cell membranes. Upon binding to the target lipid
bilayer, the protein assembles into homo-oligomeric pores of an as yet
unknown stoichiometry. Pore formation has been observed with model
liposomes consisting of phosphatidylcholine and cholesterol, but the
latter were much less susceptible to the cytolysin than were
erythrocytes or intestinal epithelial cells. We here show that liposome
permeabilization is strongly promoted if cholesterol is combined with
sphingolipids, whereby the most pronounced effects are observed with
monohexosylceramides and free ceramide. These two lipid species are
prevalent in mammalian intestinal brush border membranes.
We therefore propose that, on its natural target membranes, the
cytolysin has a dual specificity for both cholesterol and ceramides. To
assess the stoichiometry of the pore, we generated hybrid oligomers of
two naturally occurring variants of the toxin that differ in molecular
weight. On SDS-polyacrylamide gel electrophoresis, the mixed oligomers
formed a pattern of six distinct bands. Ordered by decreasing
electrophoretic mobility, the six oligomer species must comprise 0 to 5 subunits of the larger form; the pore thus is a pentamer. Due to both
lipid specificity and pore stoichiometry, V. cholerae
cytolysin represents a novel prototype in the class of bacterial
pore-forming toxins.
Vibrio cholerae O1 El Tor is the causative agent of the
current seventh cholera pandemic. The severe purging of cholera is due
to the effects of cholera enterotoxin upon the small intestine epithelial cells (1). Apart from enterotoxin, many strains of V. cholerae El Tor secrete a hemolytic toxin, V. cholerae
cytolysin (VCC).1 The latter
is also produced by most V. cholerae non-O1 strains, which
may cause intestinal and extraintestinal infections (2).
In vitro, V. cholerae cytolysin acts upon a
variety of target cells, among these are enterocytes and immune cells
(3-5). A pathogenetic role for VCC in enteritis is suggested by the
observation that the purified protein elicits fluid accumulation in
rabbit ileal loops (6, 7). Homologous cytolysins are secreted by various Aeromonas and Vibrio species that may elicit diarrheal infections and by Vibrio vulnificus, a related organism that
causes wound and septicemic infections (8).
During secretion of VCC, a signal peptide of 25 amino acids is cleaved
(9, 10). The extracellular procytolysin (79 kDa) requires further
proteolytic activation (3, 11), which can be accomplished by a variety
of proteases (12) and occurs by removal of an N-terminal fragment of 14 kDa; the latter is important for proper folding of the molecule (13).
The mature VCC of 65 kDa may undergo an additional proteolytic cleavage
close to the C terminus to yield a second active species of about 50 kDa (4).
Recently, V. cholerae cytolysin has been identified as an
oligomerizing, pore-forming protein (4, 14, 15). The process of pore
formation involves two separate steps. The water-soluble monomer first
binds reversibly to the target membrane (15). Subsequently, an unknown
number of membrane-associated monomers assemble into an oligomer that
inserts into the membrane to surround a water-filled pore of 1.5 nm
diameter (4, 16). Binding is essentially unsaturable and of low
specificity, since the cytolysin readily associates with
phosphatidylcholine liposomes (15). However, in this synthetic system,
membrane permeabilization has only been observed when
phosphatidylcholine (PC) was supplemented with cholesterol (14). Even
then, the liposomes thus obtained lag far behind animal cell membranes
in sensitivity; while rabbit erythrocytes and human enterocytes are
lysed at cytolysin concentrations of 170 pM (5, 15), a 1000 times higher amount had to be employed for permeabilization of
PC/cholesterol liposomes (14). Since V. cholerae cytolysin
does not detectably interact with rabbit erythrocyte membrane proteins
(15), we reasoned that the superior susceptibility of natural membranes
should be accounted for by lipid constituents other than cholesterol.
Characterization of these incremental lipid species should both
contribute to understanding the molecular properties of the cytolysin
and allow for improved liposome models useful in further studies on
this interesting toxin.
As an initial confirmation of our hypothesis, we found that the
susceptibility of liposomes was strongly enhanced when PC was replaced
by a crude lipid extract of bovine brain. When the constituents of this
lipid mixture were tested singly, liposome sensitization was apparent
with sphingolipids, particularly galactosylceramide, whereas only minor
effects were observed with glycerophospholipids. Comparison of various
sphingolipids indicates that the element essential for interaction with
the cytolysin consists in the common ceramide moiety.
In the second part of this study, the brain lipid liposome model was
utilized to examine the stoichiometry of the cytolysin oligomer. To
this end, we employed two naturally occurring forms of the cytolysin
that differ in molecular weight but are equally endowed with the
capability to form oligomeric pores. When these two forms were
simultaneously applied to liposomes, a mixture of oligomers was formed
that could be resolved into six distinct species by SDS-PAGE; from this
pattern, a pentameric stoichiometry of the oligomeric pore can be
inferred. The results of this study are relevant to both molecular and
pathogenetic aspects of V. cholerae cytolysin.
Bacterial Strains--
V. cholerae O1 El Tor strain
8731, which was used for isolation of the VCC 65-kDa form, was a
generous gift of Dr. R. Hall, Washington D. C. The 50-kDa form was
isolated from V. cholerae O1 El Tor strain KM 169 (4).
Purification of V. cholerae Cytolysin--
Both forms of
VCC were isolated according to published procedures. Briefly, the
65-kDa form was precipitated from culture supernatants of strain 8731 with ethanol and purified by sequential isoelectric focusing in sucrose
density gradients and hydroxyapatite chromatography (15). For the
isolation of the 50-kDa form, V. cholerae strain KM 169 was
cultured in liquid minimal medium. The cytolysin was purified by
ammonium sulfate fractionation and sequential chromatography on DE52
cellulose (Whatman), Ultrogel AcA-44 (Amersham Pharmacia Biotech), and
a Mono Q column (Amersham Pharmacia Biotech) (7).
Preparation of Liposomes--
Cholesterol,
phosphatidylethanolamine (PE), and PC from egg yolk, ceramide,
D-sphingosine, ganglioside GM1, and bovine
brain extract (composed of phosphatidylserine (PS) 10-15%, PC
15-20%, PE 20-25%, sphingomyelin (SPM) 10-15%, glycoceramides
30-40%, cholesterol 1-2%) were purchased from Fluka AG, Buchs,
Switzerland. The enumerated constituents of the brain extract were also
purchased singly in purified form (purity Calcein Release Assay--
Large unilamellar vesicles (LUV) were
produced as above, whereby the lipids after drying were resuspended in
Hepes/NaCl containing 50 mM calcein
(2',7'-bis-[N,N-bis-(carboxymethyl)aminomethyl]fluorescein). Following extrusion, the liposome suspension was passed over a column
of Sephadex G-50 (Amersham Pharmacia Biotech) equilibrated with
Hepes/NaCl to remove nonentrapped calcein. The void volume fractions
were pooled, and the lipid concentration was determined. Small
unilamellar vesicles (SUV) were prepared in an analogous manner, except
that sonication (Branson probe sonifier 250) for 6 min was substituted
for membrane extrusion. Liposomes containing 15 µg of total lipid
were incubated for 10 min at 37 °C in a volume of 100 µl with the
amounts of cytolysin indicated under "Results." Subsequently, each
sample was diluted into 3 ml of Hepes/NaCl (pH 7.5) and
immediately assayed for calcein fluorescence ( Assay of VCC Binding to Liposomes--
Liposomes varying in
composition were incubated with 1 µg of the cytolysin under the same
conditions as applied in the calcein release assay. The sample was then
supplemented with sucrose to 6% and spun in an air-driven
ultracentrifuge (Beckman Airfuge) for 15 min at 100,000 × g in order to float the liposomes. Unbound cytolysin was
sampled from below the liposome film by aspiration and quantitated by
hemolytic titration, whereas each sample was analyzed in triplicate.
2-Fold dilution series in phosphate-buffered saline, 0.05% bovine
serum albumin were prepared in a microtiter plate, and rabbit
erythrocytes were added to 1.25% final concentration. After incubation
at 37 °C for 2 h, unlysed cells were pelleted by
centrifugation. The supernatants were diluted 6-fold with
phosphate-buffered saline, and the amount of hemoglobin released was
determined photometrically at 440 nm. The hemolytic titer was read as
the highest dilution sufficient for Polyacrylamide Gel Electrophoresis (PAGE)--
SDS-PAGE was
performed according to the Laemmli method. For native PAGE, a
continuous buffer system was used containing 50 mM Tris, 50 mM glycine, and 4 mM sodium deoxycholate. Gels
were prepared with 1,4-bis(acryloyl)piperazine (Fluka) as the
cross-linker (molar ratio cross-linker/acrylamide 0.015; total
concentration of acrylamide, 5%). Electrophoresis was carried out at 5 V/cm for 1.5 h. Protein bands were visualized by silver staining
(19) or with Coomassie Blue R-250.
Cytolysin Susceptibility of Phosphatidylcholine/Cholesterol Large
Unilamellar Vesicles--
The release of calcein from liposomes
composed of PC and cholesterol has been demonstrated previously (14).
In those experiments, the cytolysin was employed at a concentration of
170 nM, which is about 1000 times more than the amount
required for lysis of rabbit erythrocytes (15). The liposomes had been
obtained by sonication, which usually results in the formation of small
unilamellar vesicles (SUV). Since the low susceptibility of the SUV
might conceivably have been related to their small size, we compared them to large unilamellar vesicles (LUV) that were prepared by extrusion through polycarbonate membranes (17). The liposomes were
incubated with various concentrations of the cytolysin (10 min,
37 °C), and the fraction of calcein released was quantitated fluorimetrically. As shown in Fig.
1A, the LUV proved even less susceptible toward the cytolysin than SUV. Since LUV are generally considered a more appropriate model of natural membranes than SUV, this
finding even accentuates the gap in susceptibility between these
artificial target membranes and natural ones.
Brain Lipids Strongly Enhance Liposome Permeabilization by
VCC--
For an initial test of the hypothesis that lipids other than
cholesterol are important in the interaction of V. cholerae
cytolysin with target membranes, we employed a crude mixture of
phospholipids and glycolipids extracted from bovine brain. These lipids
were supplemented with cholesterol to the same molar content as above (30%) and used for the preparation of LUV. Fig. 1B shows
that calcein was released from the cholesterol-enriched brain lipid liposomes at very much lower cytolysin dosages than those containing PC
(cf. Fig. 1A). Obviously, the brain extract does
contain one or more lipid species that greatly augment VCC pore formation.
We next examined the contribution of cholesterol to the sensitivity of
the brain lipid liposomes. Since the crude brain extract has a residual
cholesterol content of 1-2%, we employed a blend of purified bovine
brain lipids instead of the crude extract for the preparation of LUV
without cholesterol. PC, PE, PS, galactosylceramide, and SPM were
admixed at their respective molar fractions also prevalent in the crude
extract (see "Materials and Methods"). As seen in Fig.
1B, the cholesterol-free liposomes required a fairly high
dosage of cytolysin to yield any detectable calcein release at all, and
only very few cytolysin oligomers were detected on the liposome
membranes by SDS-PAGE. This observation reinforces the previously
established important role of cholesterol (14). On the other hand, it
may be stated that the blended brain lipid liposomes are still
similarly sensitive to VCC as are those composed of egg yolk PC and
cholesterol (cf. Fig. 1A). Thus, it appears that
both cholesterol and the incremental brain lipid species impart a low
level sensitivity to membranes when present alone, but they have a
strong cooperative effect when employed in combination.
In membrane permeabilization by VCC, monomer binding can be
distinguished from oligomerization and pore formation, and the sensitizing effect of particular membrane constituents might be related
to either of these steps. It has previously been shown that VCC
efficiently binds to membranes consisting of egg yolk PC alone (15). PC
was a major constituent of all liposome species that were employed
here. Accordingly, with all of these liposome preparations, the extent
of toxin binding ranged from 50 to 90% as assayed by hemolytic
titration. Therefore, the binding step could only account for a 2-fold
variation in membrane susceptibility, which means that the much larger
differences that were observed experimentally must mainly be due to the
oligomerization step.
Galactosylceramide Is the Major Constituent Responsible for the
Sensitivity of Brain Lipid Liposomes to VCC--
We then sought to
identify the individual lipid constituents of the crude brain extract
involved in promoting the oligomerization of VCC. To this end, the
major lipid species were obtained in pure form and incorporated into
liposomes at their respective molar fraction in the crude extract (see
"Materials and Methods"); the residual lipids were cholesterol
(molar fraction, 30%) and egg yolk PC. As an exception, bovine brain
PC was employed at 70% with no egg yolk PC. The liposomes were added
to serial dilutions of VCC. Fig. 2
displays the fraction of calcein released from the various liposome
species. The most pronounced sensitization was evidently induced by
galactosylceramide (GalCer), which was employed at 40% by mole and
approached the crude brain lipid mixture in efficacy. A slight
enhancement of susceptibility was also observed with SPM (molar content
in the membrane, 10%). In contrast, very little sensitization was
evident with any of the glycerophospholipids, with the sole exception
of bovine brain PE (membrane content, 20%) which was similar in
efficacy to SPM and clearly superior to bovine brain PC. A similar
relationship was observed with PE and PC derived from egg yolk (not
shown). The main difference between the two lipid species should
consist in their respective choline and the ethanolamine head groups;
the choline head group therefore appears to have an inhibitory effect
upon VCC pore formation.
The Ceramide Moiety Is Essential in Enhancement of VCC
Oligomerization by Sphingolipids--
In the above experiments, the
sphingolipids SPM and GalCer had performed quite differently in
sensitizing the respective liposomes to VCC. However, GalCer had been
employed to a four times higher amount, which might conceivably account
for its superior effect. For a more precise comparison of their
interaction with the cytolysin, liposomes were produced with matched
contents of SPM and GalCer. In this series of experiments, further
lipid species were also included to learn more about the importance of
various structural features of the sphingolipid molecule to the
oligomerization of VCC.
Fig. 3 displays the cytolysin dosages
required for calcein release of The 50-kDa Form of V. cholerae Cytolysin Lacks the C Terminus of
the 65-kDa Form--
In a previous report, we showed that a naturally
occurring 50-kDa form of VCC shares the N terminus of the 65-kDa form,
implying that it must have been proteolytically cleaved close to its C terminus (4). In those experiments, the 50-kDa form was only characterized by SDS-PAGE, so the possibility was not ruled out that
the proteolytic fragments remain associated under non-denaturing conditions. With the homologous V. vulnificus cytolysin, a
proteolytically nicked form has been described, the fragments of which
are held together by disulfide bonds (20). Fig.
4A shows that this is not the
case with the 50-kDa form of VCC, since identical migration was
observed under reducing and non-reducing conditions. If electrophoresis was performed in the presence of the non-denaturing detergent deoxycholate, the 50-kDa form again migrated ahead of the 65-kDa form
(Fig. 4B). This confirms that the C terminus is indeed lost upon proteolytic cleavage and is not required for cytolytic
activity.
The V. cholerae Cytolysin Pore Is a Pentamer--
The 65-kDa form
assembles into SDS-resistant oligomeric pores on suitable membranes
(14). This also holds for the 50-kDa form, whereby the two oligomer
species differ in electrophoretic mobility in good correlation with the
difference in molecular mass of the respective monomers (Fig.
5, lanes 1 and 5).
We reasoned that a mixture of the two cytolysin forms should yield
hybrid oligomers. The composition of these hybrids should be randomly distributed, and it should be reflected by their respective
electrophoretic mobility. Fig. 5 (lanes 2-4) shows that
this is indeed the case. The mixed samples formed patterns of six
evenly spaced bands, which included those representing the homogeneous
oligomers. The oligomers migrating in the lowest of these six bands
thus consisted of 50-kDa subunits only. With each of the five
subsequent bands, the number of 65-kDa subunits increased by one,
reaching a maximum of five with the topmost band. The total number of
subunits thus is always five; therefore the VCC pore is a pentamer.
When the oligomers were dissociated by boiling in SDS, their subunits
were recovered with unaltered electrophoretic mobility, which ruled out
any artifacts due to changes in covalent structure (e.g. by protease contamination; Fig. 5B).
The cytolysin of V. cholerae belongs to a homologous
family of toxins that are widespread among Vibrio and
Aeromonas species (21). Several of these are capable of
eliciting diarrheal disease, including enterotoxin-negative strains of
V. cholerae O1 El Tor (22, 23) and non-O1 serotypes (24,
25). In the latter instance, VCC most probably is pathogenetically
significant, since experimentally the purified protein imparts marked
damage to the small intestine epithelia of rabbits and mice (6, 7,
26).
The susceptibility of the epithelial cells to these cytolysins probably
reflects the adaptation of V. cholerae and
related species to the intestinal environment, but it has not yet been explained in molecular terms. The mammalian intestinal brush border membrane is distinguished by its high content of glycolipids, which may
contribute to the tubular shape of the microvilli (27, 28) and, like
phospholipids and cholesterol, amount to one-third of the total lipids.
Monohexosylceramides or ceramide represent the major glycolipids in
rats (29, 30) and humans (31). This composition of lipids very much
resembles the most susceptible synthetic liposomes characterized in the
present study. We therefore propose that its high content of
glycolipids, in conjunction with cholesterol, accounts for the
sensitivity of the brush border membrane toward V. cholerae cytolysin.
One of the most striking findings about the effect of lipids upon the
oligomerization of VCC consists in the pronounced cooperativity between
sphingolipids and cholesterol. Various sphingolipids have been reported
to associate with cholesterol in mixed membranes, which raises the
possibility that the oligomerization of VCC is mediated by
sphingolipid-cholesterol complexes rather than by the individual lipid
molecules. The interaction with cholesterol has been most thoroughly
studied with sphingomyelin. Model monolayers of sphingomyelin were
significantly condensed upon addition of cholesterol, and the sterol
was also more firmly retained by these monolayers than by ones
consisting of PC, respectively (32, 33). Similar findings have been
reported for dihexosylceramides, whereas monohexosylceramides did not
appreciably associate with cholesterol (34). To our knowledge, evidence
of stable complexes consisting of cholesterol and free ceramide is
lacking as well. Since monohexosylceramides and free ceramide are
clearly superior to SPM with respect to the enhancement of VCC
oligomerization, it appears that association of sphingolipids and
cholesterol is not essential for their interaction with the cytolysin.
Another interesting example of a protein simultaneously requiring
ceramides and cholesterol in its target lipid membrane is provided by
the E1 envelope protein of Semliki Forest virus (35, 36), which
triggers fusion of the viral envelope to the endosome membrane. Fusion,
like pore formation, requires physical separation of laterally
interacting lipid molecules, which might constitute the common
rationale behind the unusual combined specificity for two lipid
molecules, both of which are largely buried within the apolar core of
the bilayer. In line with this interpretation, both ceramide and
cholesterol are not required in binding of the cytolysin monomer but
essentially contribute in the subsequent event of oligomerization and
pore formation.
Apart from the apolar moieties of the membrane lipids, their polar head
groups also appear to play a role in the oligomerization of VCC. Of
note, a choline head group is present in both PC and sphingomyelin, and
the efficiency of oligomerization in the presence of either molecule
was clearly inferior to that observed with homologous lipid species (PE
and ceramide, respectively). The inhibitory effect of the choline head
group apparent from these results may be shared by the complex polar
tetrasaccharide moiety of the ganglioside GM1. In this
context, it should be noted that a reduction in sensitivity of rabbit
erythrocytes to VCC has been obtained by neuraminidase treatment (37).
Removal of sialic acid from the membrane glycolipids exposed terminal
galactose moieties, and susceptibility of the cells could be restored
by treating the cells with galactose oxidase. It thus appears that VCC
may interact with terminal galactosyl residues (which also occur in the
ganglioside GM1) in a non-functional manner. On the other
hand, galactosylceramide was very similar in its capability to
sensitize membranes to VCC as were glucosylceramide and free ceramide.
Possibly, the distance separating galactose from the ceramide moiety
determines whether or not oligomerization of VCC proceeds following its
binding to the sugar residue.
Sensitive model liposomes containing both cholesterol and
glycosphingolipids were utilized to assess the stoichiometry of the
V. cholerae cytolysin pore. The electrophoretic analysis of heteromers applied here to elucidate the oligomer stoichiometry was
inspired by previous work on the heptamer of Staphylococcus aureus Among the bacterial pore-formers, In sum, the present study shows that V. cholerae cytolysin
has a dual specificity for both cholesterol and ceramides, which reflects the composition of its natural target membranes and that the
pore is a pentamer. Both these properties qualify VCC as a novel
prototype within the class of bacterial pore-forming toxins, and they
probably apply to a series of homologous toxins of other Vibrionaceae
as well.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
98%) from either Sigma or
Fluka; glucosylceramide from human spleen was obtained from Sigma. The lipids were dissolved in chloroform with or without 33% methanol and
admixed at the desired molar ratios (see "Results") in a 250-ml round bottomed flask. The solvent was evaporated under a stream of
nitrogen, and the lipid film was dried for 30 min under vacuum. Following resuspension of the lipids in Hepes/NaCl to 5 mg/ml, large
unilamellar vesicles (LUV) were formed by repeatedly extruding the
suspension through polycarbonate membranes (Nuclepore, CA; 100-nm pore
size) (17), whereby the extrusion apparatus (Lipex Biomembranes,
Vancouver, Canada) was thermostatted at 40 or 50 °C if required. The
lipid concentration in the final sample was determined using a
commercial enzymatic cholesterol assay (Boehringer Mannheim). If the
liposomes did not contain cholesterol, phospholipids were quantitated
by phosphorus analysis (18).
ex, 488 nm,
em, 520 nm) in a SPEX Fluoromax fluorimeter.
The fraction of calcein released was calculated from the increase of
fluorescence over that of a control sample incubated without cytolysin,
whereas the fluorescence maximum corresponding to 100% release was
determined on a sample solubilized with sodium deoxycholate (final
concentration, 6 mM).
60% hemolysis.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
VCC-mediated release of calcein from
liposomes differing in size or composition. Liposomes with
entrapped calcein were added to a dilution series of V. cholerae cytolysin (final concentration of lipid, 150 µg/ml).
After 10 min at 37 °C, the calcein released was quantitated
fluorimetrically; 100% release corresponds to the fluorescence of a
detergent-lysed sample. A, SUV and LUV consisting of
phosphatidylcholine (molar content, 70%) and cholesterol (30%).
B, LUV prepared from crude bovine brain lipids, supplemented
with cholesterol to a molar content of 30% or from a corresponding
mixture of purified brain lipids lacking cholesterol. Inset,
SDS-PAGE of VCC incubated with liposomes. The upper
bands correspond to the oligomer. Left, brain lipid LUV
with cholesterol; right, brain lipid LUV without
cholesterol.
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Fig. 2.
The effect of individual brain lipid species
upon membrane permeabilization by VCC. The major components of
bovine brain lipids were incorporated to the molar ratios indicated
into liposomes, whereby the residual lipids were made up by cholesterol
(30%) and egg yolk phosphatidylcholine. Membrane permeabilization was
assayed by release of calcein as detailed in the legend to Fig. 1.
GalCer, SPM, PE, and PS were employed at their respective molar ratios
also prevalent in the crude brain extract. Brain PC (which amounts to
15% in the extract) was used at 70% without any egg yolk PC.
50% from liposomes containing 10, 20, or 40% of the respective sphingolipids (missing values for 20 or
40% indicate that homogeneous and stable liposome preparations could
not be obtained under our experimental conditions.) The essential
findings can be stated as follows: SPM is clearly inferior to GalCer
also if both are employed at equivalent amounts. The ganglioside
GM1 (which possesses a tetrasaccharide head group attached
to ceramide) is similar in efficacy to SPM. Both glucosylceramide and
free ceramide closely match GalCer in their degree of liposome
sensitization, which indicates that ceramide does not require any head
group to be attached. Sphingosine, however, is only weakly effective, indicating that removal of the amide-linked fatty acid from the ceramide molecule impairs its ability to support VCC oligomerization. We conclude that the ceramide moiety is crucial in the interaction of
sphingolipids with VCC.
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Fig. 3.
The effect of sphingolipid structure and
concentration upon liposome permeabilization by VCC. A series of
different sphingolipids were incorporated into liposomes to a molar
content of 10, 20, or 40%, whereas the rest of the lipid mixture
consisted of cholesterol (30%) and egg yolk PC. Stable liposome
preparations could not be obtained with 40% SPM and 20 or 40%
sphingosine, respectively. Membrane permeabilization was assayed as
detailed in the legend to Fig. 1; bars indicate the lowest
concentration of VCC sufficient for release of calcein from the
liposomes to 50%.
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Fig. 4.
Denaturing and non-denaturing PAGE of two
forms of V. cholerae cytolysin isolated from two
different strains. A, SDS-PAGE. Lane 1, size
standard; lane 2, 65-kDa form of VCC, reduced with
1,4-dithiothreitol; lane 3, 65-kDa form, not reduced;
lane 4, 50-kDa form, reduced; lane 5, 50-kDa
form, not reduced. The 50-kDa form lacks a C-terminal (4) fragment of
about 14 kDa. Loss of this fragment does not require reduction of
disulfide bonds. B, non-denaturing PAGE with sodium
deoxycholate. Lane 1, 65-kDa form; lane 2, 50-kDa
form. The two forms exhibit a similar difference in mobility as above,
indicating that the 50-kDa form is lacking the C-terminal fragment
under native conditions, too.
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Fig. 5.
Characterization of hybrid V. cholerae
cytolysin oligomers by SDS-PAGE (silver staining). The
oligomers of the cytolysin resist dissociation by SDS at room
temperature (15). The two cytolysin forms of 65 and 50 kDa were admixed
at varying ratios. From the mixtures, oligomers were generated by
incubation with brain lipid/cholesterol liposomes at 37 °C.
A, the samples were applied to SDS-PAGE without prior heat
treatment. The following ratios (65:50 kDa) were used: lane
1, 100:0; lane 2, 90:10; lane 3, 70:30;
lane 4, 40:60; lane 5, 0:100. B, the
samples correspond to those in A but were dissociated at
95 °C prior to electrophoresis.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-hemolysin (38). In the work cited, the two toxin
variants required were obtained by chemical modification of single
cysteine mutants. Where chemical modification or limited proteolysis
are inappropriate, extending or truncating the termini of a protein molecule at the DNA level might be used to produce the variant species
for heteromer analysis. The latter method should thus be more generally
useful to determine the stoichiometries of toxin pores and, if combined
with non-denaturing analytical separation, other homo-oligomeric proteins.
-hemolysin provides the only
example of an oligomer structure determined at high resolution (39).
Its transmembrane portion consists of a
-barrel with 7-fold
rotational symmetry. Heptameric stoichiometry and
-barrel structure
has also been confirmed with the protective antigen component of
anthrax toxin (40, 41). As a pentamer, VCC so far is unique within this
particular class of toxins, but there are several previous examples of
pentameric transmembrane channels in general. One of those is provided
by the B subunit oligomer of V. cholerae enteroxin. The
hollow center of the latter is lined by five
-helices (42). Helical
structure has also been suggested for the intramembranous portion of
the nicotinic acetylcholine receptor (43) and for the cardiac calcium
channel phospholamban (44). It would be most interesting to determine
which one of the two above structural paradigms applies to the
transmembrane part of the VCC pore.
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FOOTNOTES |
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* This study was supported by the Deutsche Forschungsgemeinschaft Grant SFB 311.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.
Submitted part of this work as M.D. thesis.
§ To whom correspondence should be addressed.
The abbreviations used are: VCC, V. cholerae cytolysin; GalCer, galactosylceramide; LUV, large unilamellar vesicles; PE, phosphatidylethanolamine; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; SPM, sphingomyelin; SUV, small unilamellar vesicles.
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