Influence of Gb3 glycosphingolipids differing in their fatty acid chain on the phase behaviour of solid supported membranes: Chemical syntheses and impact of Shiga toxin binding

Article abstract of DOI:10.1039/c4sc01290a

The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb₃. In biological membranes, Gb₃ glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb₃ complex. To analyse the binding of STxB to different Gb₃s, we chemically synthesized saturated, unsaturated, α-hydroxylated Gb₃s and a combination thereof, all based on a C₂₄-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb₃s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescence- and atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb ₃ significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb₃. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb₃s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb₃s. Large interphase lipid redistribution is observed for α-hydroxylated Gb₃ doped membranes. Our results clearly demonstrate that the fatty acid of Gb₃ strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes. the Partner Organisations 2014.

Full text of DOI:10.1039/c4sc01290a

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Influence of Gb3 glycosphingolipids differing in
their fatty acid chain on the phase behaviour of
solid supported membranes: chemical syntheses
and impact of Shiga toxin binding†
Cite this: Chem. Sci., 2014, 5, 3104
Ole M. Schutte,‡a Annika Ries,‡a Alexander Orth,a Lukas J. Patalag,ab
¨
¨
a
b
Winfried Romer,cd Claudia Steinem and Daniel B. Werz
*
*
The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga
holotoxin, binds specifically to the glycosphingolipid Gb3. In biological membranes, Gb3 glycosphingolipids
differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding
behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb3 complex. To analyse the
binding of STxB to different Gb3s, we chemically synthesized saturated, unsaturated, a-hydroxylated Gb3s
and a combination thereof, all based on a C24-fatty acid chain starting from monosaccharide building
blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb3s were inserted into
solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple
mimetic of the outer leaflet of animal cell membranes. By fluorescence- and atomic force microscopy the
phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty
acid of Gb3 significantly alters the ratio between the ordered and disordered phase and induces a third
intermediate phase in the presence of unsaturated Gb3. The lateral organization of STxB on the
membranes varies significantly. While STxB attached to membranes with Gb3s with saturated fatty acids
forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb3s. Large
interphase lipid redistribution is observed for a-hydroxylated Gb3 doped membranes. Our results clearly
demonstrate that the fatty acid of Gb3 strongly influences the lateral organization of STxB on the
membrane and impacts the overall membrane organization of phase-separated lipid membranes.
Received 5th May 2014
Accepted 13th May 2014
DOI: 10.1039/c4sc01290a
www.rsc.org/chemicalscience
Cholera toxin tightly attach to the plasma membrane via the
ganglioside GM1 resulting in plasma membrane invagina-
tions.1,2 Shiga toxin, an AB5 protein produced by Shigella dys-
Introduction
Glycosphingolipids are the cellular receptors of a number of
viruses and bacterial toxins. For example, simian virus 40 and
other polyoma viruses as well as the bacterial AB5 protein
enteriae and by enterohemorrhagic strains of Escherichia coli
(EHEC) binds via its homopentameric B-subunits (STxB) to the
neutral globoside Gb3.3,4 This process is the initial step for Shiga
toxin to efficiently enter cells. Aer endocytosis, Shiga toxin
leaves the endocytic pathway at the level of early endosomes
aInstitute of Organic and Biomolecular Chemistry, University of Gottingen,
¨
¨
Tammannstr. 2, 37077 Gottingen, Germany. E-mail: csteine@gwdg.de
5
6
¨
following the retrograde transport route. Romer et al. showed
that the pentameric B-subunits, which can bind up to 15 Gb3
molecules, induce a clathrin- and caveolin-independent tubule
formation upon attachment to HeLa-cells and that invagina-
tions were even found in articial membranes, when STxB
binds to giant unilamellar vesicles (GUVs). These results
suggest a signicant lipid contribution to the earliest steps of
toxin uptake into cells and there are several lines of evidence
that the cytotoxicity of Shiga toxin is strongly associated with
Gb3 density and binding to cholesterol-containing lipid ras.5,7,8
Following this hypothesis, it has been demonstrated that
STxB binding leads to an increased lipid order in cellular
membranes.6 Results obtained from articial membranes on
b
¨
Institute of Organic Chemistry, Technische Universitat Braunschweig, Hagenring 30,
38106 Braunschweig, Germany. E-mail: d.werz@tu-braunschweig.de
cBIOSS – Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg,
¨
Schanzlestrabe 18, 79104 Freiburg, Germany. E-mail: winfried.roemer@bioss.
uni-freiburg.de
d
¨
Institute of Biology II, Albert-Ludwigs-University Freiburg, Schanzlestraße 1, 79104
Freiburg, Germany
† Electronic supplementary information (ESI) available: Experimental details of
the synthesis and analytical data of 27–30; detailed experimental procedures on
the preparation and analysis of solid supported bilayers; fatty acid distribution
of natural Gb3 and bovine brain SM; additional uorescence and AFM images
of phase-separated lipid bilayers; calculation of STxB surface coverage and
receptor coverage; ternary phase diagram to estimate phase compositions. See
DOI: 10.1039/c4sc01290a
‡ These authors contributed equally to this work.
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solid substrates as well as from pore-spanning membranes
further showed that STxB is capable of reorganizing lipids
within these membranes concomitant with protein cluster
formation.9,10 This process can induce a protein induced local
compaction of particular lipids in one leaet leading to an
asymmetric reduction in membrane area, which is prerequisite
for membrane invaginations.11
Results
Synthesis of Gb3 glycosphingolipids
The power of
a synthetic approach towards Gb3 glyco-
sphingolipids is most promising due to the highly dened
nature of the obtained material. From a retrosynthetic point of
view a glycosphingolipid can be divided into three major parts:
(i) the carbohydrate head group as the hydrophilic and the
ceramide as the lipophilic part that can be further subdivided
into (ii) a sphingosine such as D-erythro-sphingosine and (iii) a
fatty acid being attached to the amine of the sphingosine
(Scheme 1). We assembled four different glycosphingolipids,
which consist of the globotriaose head group, the sphingosine,
but differ in the fatty acid chain. Saturated, unsaturated,
a-hydroxylated derivatives and a combination thereof were
prepared, all based on a C24 fatty acid. C24 fatty acids have been
chosen as they are the major constituent (>50%, ESI†) found in
natural Gb3 mixtures such as toxin insensitive erythrocytes,14
HeLa-cells,24 and HEp-2 cells.25
The globotriaoside is traced back to three different mono-
saccharide building blocks (Scheme 1, 1–3). Glucose derivative 1
was obtained in four steps starting from 3,6-di-O-benzyl-D-glu-
cal26 being an ideal precursor for various glucose-derived
building blocks. Fmoc protection of the 4-O-hydroxyl group
followed by epoxidation with dimethyldioxirane (DMDO)27 and
epoxide opening using benzylic alcohol led to a glucose deriv-
ative with a free hydroxyl group in C-2 position. Acetylation and
Fmoc removal provided glucosyl acceptor 1 (ESI†). Galactosyl
building block 2 was synthesized starting from 3,6-di-O-benzyl-
D-galactal (10).28 Fmoc protection of the free hydroxyl and
subsequent epoxidation with DMDO followed by epoxide
opening with dibutyl phosphate terminated by pivaloyl protec-
tion of the emerging 2-hydroxyl afforded building block 2
(ESI†).29 The third unit, an a-linked galactose, was prepared
In all these studies, mixtures of Gb3 from natural sources have
been used, which are composed of Gb3 molecules with different
fatty acid compositions. Even though the Gb3-concentration is
only a few mol% in these membranes, there is strong evidence
that the fatty acid composition of Gb3 signicantly inuences the
process of Shiga toxin binding,12 membrane organization,9,13
invagination,6 and scission.14 Lingwood and coworkers found
that Gb3s with long fatty acid chains (C20–C24) bind Shiga toxin
with higher affinity than those with short fatty acid chains (C12,
C14).15 Also, the a-hydroxylation of the fatty acid of Gb3 gave rise
to a larger binding affinity and larger binding capacity of STxB
compared to the non-hydroxylated fatty acid.13 Besides the
observed differences in the STxB binding behaviour, a-hydroxy-
lated fatty acids attached to glycolipids have also been shown to
strongly inuence local membrane organization owing to their
ability to form hydrogen bonds.16–18 However, as yet there is no
systematic study available addressing the impact of the fatty acid
composition of Gb3 on the organization of lipid membranes that
partially mimic the composition of the outer leaet of animal
plasma membranes. This arises from the fact that, apart from
some reports on semisynthetic approaches to generate Gb3
molecules,15,19 chemical access to these highly complex glyco-
lipids is a challenging and time-consuming endeavour. Never-
theless, such a synthetic approach is the method of choice to
gain chemically pure material which is needed for proper
biophysical investigations. In 2007, Florent and co-workers
already devised a synthetic route from the trisaccharidic subunit
to Gb3-C22:0 and Gb3-C22:1 glycosphingolipids.6
In this paper, we describe a generally applicable and
modular strategy to assemble the target glycosphingolipids
starting from simple monosaccharide building blocks,
sphingosine and fatty acids. Saturated, unsaturated,
a-hydroxylated fatty acids and
a combination thereof
were employed. These molecules were reconstituted into
membranes composed of sphingomyelin, cholesterol, and
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) resembling
the composition of the outer leaet of animal plasma
membranes and that are known to phase-separate.20,21 Solid
supported membranes (SSMs) in conjunction with uores-
cence microscopy and atomic force microscopy were used to
investigate the lateral organization of the membranes as a
function of Gb3 composition and STxB binding. Despite a
reduced lateral lipid mobility found in SSMs, these
membranes are sufficiently dynamic to allow for lipid rear-
rangement while offering the possibility to use uorescence
microscopy and high resolution atomic force microscopy
(AFM) to monitor nanometre-sized domains.22,23 We provide
evidence that the fatty acid strongly inuences the phase
behaviour of the membranes as well as the mode of STxB
cluster formation.
Scheme 1 Retrosynthetic analysis of Gb3 glycosphingolipids.
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trisaccharide 14 in 79% yield (over three steps) and pure a-
selectivity as it was anticipated by the remote participation30 of
the benzoyl protecting group at C-4. In addition, a reaction
temperature of ꢀ60 ꢁC proved to be benecial as well to achieve
complete diastereoselectivity. In case of the more reactive per-
benzylated galactosyl phosphate both, the yield and the selec-
tivity, dramatically dropped down.
The commonly used technique to deprotect the permanent
benzyl protecting groups by hydrogenolysis at the very end of an
oligosaccharide synthesis is not a choice in the assembly of
glycosphingolipids since their double bond would be destroyed
during such a reduction step. Thus, the protecting groups of the
globotriaose unit had to be exchanged before the union with the
lipid part takes place. Taking these difficulties into account,
benzoyl groups being easily cleaved under basic conditions were
installed. In order to get perbenzoylated material, rstly, the
three different acyl groups were removed under strong basic
conditions, followed by a hydrogenolysis of the benzyl ethers
using Pearlman's catalyst. Perbenzoylation of the naked trisac-
charide was performed followed by removal of the anomeric
benzoate using methylamine. Finally, a trichloroacetimidate
moiety was installed as leaving group to yield globotriaoside
derivative 17 in 99% yield.
In addition, two hydroxylated fatty acids being not
commercially available had to be prepared. Based on a route
developed by Kiso, Ando and co-workers, a-hydroxylated tetra-
cosanoic acid 22 was generated (Scheme 4a).32 Alkyne 18, readily
available from 1-eicosanol, was used to open epoxide 19. The
secondary hydroxyl group was directly protected with a benzoyl
group to afford 20 in 45% yield over two steps. Hydrogenation
using palladium on charcoal furnished the saturated debenzy-
lated alkane chain whereas Pearlman's catalyst induced an
acyl group migration. Finally, the primary hydroxyl group of 21
was oxidized to fatty acid 22 in 73% yield using TEMPO/TCCA
Scheme 2 Preparation of a-galactosyl building block 3.
starting from 10 as well (Scheme 2). To facilitate the formation
of the a-galactosidic linkage in the trisaccharide a remote-
participating group (Bz)30 was installed at the 4-hydroxyl of 10.
DMDO-mediated epoxidation of galactal 11 and attack with
p-methoxyphenol (MPOH) led to acetal 12 in 90% yield. Benzyl
ether formation and removal of the anomeric protecting group
under oxidative conditions furnished hemiacetal 13.
The latter was rst converted into a trichloroacetimidate31
and subsequently transformed to phosphate 3 which proved to
be more suitable in this glycosylation reaction than the tri-
chloroacetimidate analogue.
With these monosaccharide building blocks in hand we
started the assembly of the globotriaose. The sequence
commences with the union of building blocks 1 and 2 using
TMSOTf as Lewis acidic promoter (Scheme 3). The b-selectivity
is ensured due to neighbouring group participation of the
pivaloate of galactose building block 2. Subsequent removal of
the Fmoc protecting group under mild basic conditions fol-
lowed by the second glycosylation with building block 3 led to
Scheme 3 Assembly of the globotriaosyl trichloroacetimidate 17.
3106 | Chem. Sci., 2014, 5, 3104–3114
Scheme 4 Syntheses of hydroxylated fatty acids 22 and 24.
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conditions. The protected a-hydroxylated nervonic acid 24 established lipid mixture DOPC/sphingomyelin (SM)/choles-
was synthesized starting from nervonic acid in two steps terol (Chol) in a molar ratio of 2 : 2 : 1 on mica surfaces.36
(Scheme 4b).
Vesicle spreading was achieved at elevated temperatures (55 ꢁC)
Aer carboxylate enolate formation using freshly prepared above the phase-transition temperature of the lipid mixture
LDA, a dry oxygen atmosphere attached to the reaction, deri- (Tm z 40 C).37 At room temperature, the membrane is phase-
ꢁ
vatised the a-position with a hydroxyl group to yield 9 in 56%.33 separated into a lo and a ld phase, as can be revealed from
Final benzoylation of this secondary hydroxyl gave the desired uorescence micrographs (ESI, Fig. S1A†) using sterically
fatty acid 24. To complete total synthesis, globotriaosyl tri- demanding uorophors (Bodipy-PC, Texas Red DHPE, Oregon
chloroacetimidate 17 and the protected sphingosine 4 or 5,34 Green DHPE) that preferentially partition into the ld phase.38
respectively, were coupled in a glycosylation reaction using The overall membrane integrity was veried for each experi-
BF3$OEt2 to afford 25 or 26, in moderate yields (Scheme 5). In ment using the dye perylene, which distributes more equally
case of the benzoyl protected sphingosine only a minor acyl between the lo and ld phase (ESI, Fig. S1B†). The percentage
group migration was observed. Staudinger reduction of the of the lo phase was determined by pixel analysis to be 66 ꢂ 6%
azide and direct coupling with the fatty acids 6–9 under peptide (n ¼ 35). To this lipid mixture, the globotriaosylceramide Gb3
coupling conditions (e.g. HATU or HOBt/EDCI)35 led to four serving as the receptor lipid for STxB was added by replacing
protected glycosphingolipids, varying in their acyl chains. 5 mol% of the used bovine brain SM (ESI, Table S2†). This
´
Global deprotection under Zemplen conditions resulted in the exchange retains the principal lo/ld phase-separation of the
desired glycosphingolipids 27–30.
membranes.9 To scrutinize the inuence of the fatty acid of Gb3
on the phase behaviour of the SSMs, one of the four synthesized
Gb3 molecules (27–30) were incorporated into the phase-sepa-
rated membranes. Besides the Gb3 with the C24:0 fatty acid (27,
Gb3-C24:0), we analysed the inuence of the a-hydroxylated
fatty acid (29, Gb3-C24:0-2OH), an unsaturated fatty acid (28,
Gb3-C24:1), as well as both, an unsaturated and a-hydroxylated
fatty acid (30, Gb3-C24:1-2OH).
Phase behaviour of solid supported membranes doped with
different Gb3s
SSMs that separate into a liquid-ordered (lo) and liquid-disor-
dered (ld) phase at room temperature were obtained by
spreading unilamellar vesicles composed of the well-
Characteristic uorescence images of a solid supported
membrane composed of DOPC/SM/Chol/Gb3 (40 : 35 : 20 : 5)
Fig.
1
SSMs
composed
of
DOPC/SM/Chol/Gb3-C24:0
(40 : 35 : 20 : 5) doped with perylene (0.5 mol%) and Texas Red DHPE
(0.1 mol%) on mica. (A) Fluorescence micrograph of Texas Red DHPE
(false coloured in green). Membranes segregate into liquid-ordered
(lo, dark) and liquid-disordered domains (ld, green). Scale bar: 20 mm.
(B) Magnification of the region marked in (A). Scale bar: 10 mm. (C) AFM
image showing a higher lo phase and a lower ld phase (darker colour
coding). Scale bar: 5 mm. (D) Histogram analysis showing a height
difference Dh(lo/ld) ¼ 0.7 ꢂ 0.2 nm for the image shown in (C).
Scheme 5 Gb3 glycosphingolipid assembly.
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with Gb3-C24:0 are shown in Fig. 1A and B. The uorescence of
Texas Red DHPE (false coloured in green) indicates the ld phase,
while the dark areas are attributed to the lo phase, which
excludes the uorescent lipid. The area percentage of the lo
phase was determined by pixel analysis and amounts to 74 ꢂ
7% (n ¼ 84). By means of atomic force microscopy (AFM), the
topography of the phase-separated membranes was analysed
(Fig. 1C). Domains of lower height are visualized in the AFM
image, which are assigned to the ld phase (darker colour coding)
embedded in a higher lo phase. The height difference between
the lo and ld phase was determined by histogram analysis
(Fig. 1D) to be Dh(lo/ld) ¼ 0.6 ꢂ 0.2 nm (n ¼ 21) in agreement
with height differences reported in literature.39
For membranes containing 5 mol% of Gb3-C24:0-2OH (ESI,
Fig. S2†), again a phase-separated lipid membrane was visual-
ized in uorescence microscopy as well as in atomic force
microscopy, from which the lo and ld phase could be unam-
biguously assigned with a typical height difference of Dh(lo/ld) ¼
0.8 ꢂ 0.1 nm (n ¼ 87). However, the area percentage of the lo
phase was, with 56 ꢂ 5% (n ¼ 56), signicantly lower compared
to the lipid mixture containing 5 mol% of Gb3-C24:0. Given the
fact that only the 5 mol% component Gb3 was replaced in the
lipid mixture, while all other lipid components remained
constant, a change of about 20% of the area percentage of the lo
phase was rather unexpected suggesting a more global impact
of the Gb3 molecules on the phase-separation behaviour of the
membrane as discussed in more detail below.
Fig.
2
SSMs
composed
of
DOPC/SM/Chol/Gb3-C24:1
(40 : 35 : 20 : 5) doped with perylene (0.5 mol%) and Texas Red DHPE
(0.1 mol%). (A) Fluorescence micrograph of Texas Red DHPE (false
coloured in green). Membranes segregate into three phases as indi-
cated by three brightness levels. Scale bar: 10 mm. (B) Magnification of
the region marked in (A). Additionally to liquid-ordered (lo, dark) and
liquid-disordered domains (ld, bright green) a third lipid phase of
intermediate brightness (li) is identified. Scale bar: 4 mm. (C) AFM image
showing the three phases. While the lowest phase is assigned to the ld
phase, the two other phases differing in height cannot be unambig-
uously assigned to the lo and li phase. Scale bar: 2 mm. (D) Histogram
analysis showing the heights of the three phases. Taking the ld phase as
the lowest phase, two height differences of Dh1 ¼ 0.9 ꢂ 0.4 nm and
Dh2 ¼ 1.8 ꢂ 0.4 nm are calculated for the image shown in (C).
Surprisingly different phase behaviour was observed if the
lipid mixture contained Gb3-C24:1. The uorescence micro-
graphs of Texas Red DHPE show three uorescence intensities
(Fig. 2A and B). While a bright green phase is still clearly visible
and can be identied by the enrichment of the uorescent dye, a
dark phase as well as a phase with an intermediate brightness is
observed. We assign the membrane phase, from which Texas
Red DHPE is mostly excluded to the lo phase, while the phase
with the highest uorescence intensity is attributed to the ld
phase. The phase of intermediate brightness is termed li phase
and was found independently of the ld marker dye used (ESI†).
According to this assignment, one can determine the area
percentages of all three phases (n ¼ 73) being for the ld phase
23 ꢂ 6%, for the li phase 36 ꢂ 8% and for the lo phase 40 ꢂ 6%.
By atomic force microscopy, only in rare cases the three phases
were resolved by their differences in height (Fig. 2C). In case of
the detection of three different height levels we assigned the
lowest one to the ld phase. As it is not possible to assign the li
phase and the lo phase to the different heights in the AFM
images, we took the ld phase as the reference and determined
two height differences with Dh1 ¼ 1.2 ꢂ 0.2 nm (n ¼ 4) and
Dh2 ¼ 1.6 ꢂ 0.2 nm (n ¼ 5). In several AFM images, the two
different heights could not be resolved resulting in an averaged
value of Dh1/2 ¼ 1.2 ꢂ 0.2 (n ¼ 68).
Table 1 Ratios of the different membrane phases (lo: liquid-ordered, li:
liquid-intermediate) in DOPC/SM/Chol/Gb3 (40 : 35 : 20 : 5) bilayers
doped with different Gb3s on mica and height differences observed in
AFM images. The ratios were determined by pixel analysis using fluo-
rescence images. Height differences were extracted by fitting Gauss-
ians to the height histograms. For compound 27, 29, and 30 Dh2
corresponds to the height difference between lo and ld phase (Dh(lo/ld))
(number of analysed fluorescence images and height histograms is
given in brackets)
Compound
lo/%
li/%
Dh2/nm
Dh1/nm
27
28
29
30
74 ꢂ 7 (84)
40 ꢂ 6 (73)
56 ꢂ 5 (56)
70 ꢂ 7 (51)
—
0.6 ꢂ 0.2 (21)
1.6 ꢂ 0.2 (5)
0.8 ꢂ 0.1 (87)
0.5 ꢂ 0.2 (31)
—
36 ꢂ 8
1.2 ꢂ 0.2 (4)
—
—
—
—
well as the height differences between the observed lipid phases
For Gb3-C24:1-2OH, which contains the cis-double bond as as deduced from AFM analysis in the otherwise identical lipid
well as the hydroxylation in the 2-position, the membrane again mixtures.
only separated into two phases assigned to as ld and lo with an
Our ndings demonstrate that the fatty acid composition of
area percentage of 70 ꢂ 7% (n ¼ 51) resembling the situation of the glycosphingolipid Gb3 strongly inuences the phase
Gb3-C24:0 more than the other Gb3 species. Table 1 summarizes behaviour of the phase-separated lipid membranes, even
the area percentages found for the different Gb3 molecules as though it amounts only to 5 mol% of the whole lipid
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composition. This result implies that Gb3 strongly interacts specic binding of STxB to DOPC/SM/Chol (2 : 2 : 1)
with the other lipid components thus altering the phase membranes lacking Gb3 can be neglected.
behaviour.
For Gb3-C24:0 containing membranes, the Oregon Green
DHPE micrographs show phase-separated membranes with lo
phase and ld phase aer STxB binding (Fig. 3A). The corre-
sponding Cy3-uorescence image of the bound STxB shows
inverse uorescence, indicating that the protein has been
bound to the lo phase, in which the Gb3 molecules are located
(Fig. 3B). The area percentage of the lo phase does not change
upon protein binding (76 ꢂ 11%, n ¼ 52). From the STxB-Cy3
uorescence image as well as from the AFM images, it becomes
obvious that the protein does not homogeneously bind to the lo
phase but forms clusters. The height difference between the
protein clusters on the lo phase and the overall height of
protein-decorated lo-phase is Dh(lo+STxB_clusters/lo+STxB) ¼ 0.8 ꢂ
0.1 nm (n ¼ 6). The overall increase in height of the lo phase in
the presence of bound protein is only very minor with an
increase of Dh(lo+STxB/ld) ꢀ Dh(lo/ld) ¼ 0.1 ꢂ 0.4 nm indicating
that the protein surface coverage on the entire lo phase is, except
for the dense protein domains, rather low.
STxB binding to solid supported membranes doped with
different Gb3s
The B-subunits of the protein Shiga toxin are known to alter the
phase behaviour of membranes, when they bind to their
receptor lipid Gb3.9,10,40 We thus elucidated the impact of STxB
binding on the phase behaviour of the lipid mixture containing
different Gb3 species by using uorescently labelled STxB. In
lo/ld phase-separated membranes containing a mixture of
different Gb3 species, it has been shown that the Gb3s are
located in the lo phase aer STxB binding.9 The position of the
protein uorescence therefore allows us to localize the Gb3
molecules aer protein binding. Aer preparation of SSMs
composed of DOPC/SM/Chol/Gb3 (40 : 35 : 20 : 5) on mica
containing one of the four different Gb3 species, they were
incubated with 60 nM of STxB for 1 h, rinsed with buffer and
inspected by uorescence microscopy. The uorescent lipid
Texas Red DHPE was exchanged against Oregon Green DHPE or
Bodipy-PC, which also label the ld phase, to be able to monitor
the ld phase (green) at the same time as the uorescence of the
Cy3-labeled STxB (red). Control experiments reveal that non-
Phase-separated membranes containing Gb3-C24:0-2OH
with considerably reduced area percentage of the lo phase prior
to protein binding of only 56 ꢂ 5%, experience a signicant
increase in lo phase aer STxB binding (77 ꢂ 5%, n ¼ 62).
The STxB-Cy3 uorescence is inhomogeneous (ESI, Fig. S3†)
Fig.
3
SSMs
composed
of
DOPC/SM/Chol/Gb3-C24:0 Fig. 4 SSMs composed of DOPC/SM/Chol/Gb3-C24:1 (40 : 35 : 20 : 5)
(40 : 35 : 20 : 5) doped with perylene (0.5 mol%) and Oregon Green doped with perylene (0.5 mol%) and Bodipy-PC (0.1 mol%) after incu-
DHPE (0.1 mol%) after incubation with 60 nM Cy3-labeled STxB. (A) bation with 60 nM Cy3-labeled STxB. (A) Fluorescence micrograph of
Fluorescence micrograph of Oregon Green DHPE. Membranes Bodipy-PC. The membrane segregates into three phases as indicated by
segregate into lo (dark) and ld (green) domains. Scale bar: 15 mm. (B) three brightness levels. Scale bar: 15 mm. (B) Fluorescence of Cy3-
Fluorescence micrograph of STxB-Cy3. The inhomogeneous red labeled STxB shows protein binding to the lo phase. Top right corner:
fluorescence of the labeled protein is inverse to the Oregon Green overlay with Bodipy-PC fluorescence. Scale bar: 15 mm. (C) AFM image
DHPE labeled ld phase. Scale bar: 15 mm. (C) AFM image showing a of the bilayer after STxB incubation. Three different height levels are
lower ld phase (dark colour coding), higher lo phase and STxB clusters observed. Scale bar: 2 mm. (D) Histogram analysis of the heights of the
on top of the ordered phase. Scale bar: 2 mm. (D) Height difference three phases. Taking the ld phase as the lowest phase, where no protein
between STxB clusters and protein decorated lo phase is determined has been bound to, two height differences of Dh(li/ld) ¼ 1.2 ꢂ 0.8 nm and
to Dh(lo+STxB_clusters/lo+STxB) ¼ 0.6 ꢂ 0.2 nm for the image shown in (C). Dh(lo+STxB/ld) ¼ 2.4 ꢂ 0.8 nm are calculated.
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namely to the assigned lo phase. This in turn would imply that
aer STxB binding, the Gb3 receptors are located only in the lo
phase. The area percentages of the phases do not change upon
STxB binding being 43 ꢂ 7% and 31 ꢂ 8% for the lo and li phase,
respectively. However, in contrast to the other Gb3 containing
membranes, the domain morphology changes considerably, i.e.
all domains condense into larger ones. The height difference of
Dh(li/ld) ¼ 1.3 ꢂ 0.7 nm (n ¼ 12) corresponds to the height
difference between the ld phase and the non-protein covered li
phase. The rather large difference between the ld and li phase
might be interpreted in a sense that the li phase is highly
ordered, composed of mostly saturated lipids. The protein
bound to the Gb3 containing lo phase results in a height
difference of Dh(lo+STxB/ld) ¼ 2.6 ꢂ 0.6 nm (n ¼ 26).
For membranes containing Gb3-C24:1-2OH, two phases are
identied with the lo phase being visualized by the red STxB-Cy3
uorescence (Fig. 5A and B). Of note, in accordance with the
observation made for the saturated, a-hydroxylated compound
Gb3-C24:0-2OH, the area fraction of the lo phase is signicantly
increased from 70 ꢂ 7% to 87 ꢂ 7% (n ¼ 42).
Fig.
5 SSMs composed of DOPC/SM/Chol/Gb3-C24:1-2OH
This result implies that more DOPC molecules are recruited
into the lipid dye excluding phase. In contrast to the other Gb3
derivatives under investigation, the topography mapped by AFM
(Fig. 5C) only shows two height levels with a height difference of
Dh(lo+STxB/ld) ¼ 2.7 ꢂ 0.5 nm (n ¼ 44).
(40 : 35 : 20 : 5) doped with perylene (0.5 mol%) and Oregon Green
DHPE (0.1 mol%) after incubation with 60 nM Cy3-labeled STxB. (A)
Fluorescence micrograph of Oregon Green DHPE. The membrane
segregates into a lo (dark) and ld (green) phase. Scale bar: 15 mm. (B)
Fluorescence micrograph of the STxB-Cy3 fluorescence. STxB is only
located on the lo domains. Scale bar: 15 mm. (C) AFM image showing
the higher lo phase and a lower ld phase (darker colour coding). Scale
bar: 2 mm. (D) Height difference between the two identified phases in
the histogram with Dh(lo+STxB/ld) ¼ 2.3 ꢂ 0.5 nm.
The higher phase is the lo phase with bound protein as
deduced from the uorescence images. No individual proteins
can be resolved, however the increase in height as a result of
protein binding amounts to hSTxB ¼ Dh(lo+STxB/ld) ꢀ Dh(lo/ld) ¼
2.2 ꢂ 0.7 nm, which indicates a high protein density on the
surface resulting in a thickness that matches the crystallo-
graphic dimensions of STxB.41
Table 2 summarizes the properties observed in uorescence
microscopy and atomic force microscopy aer STxB binding to
the membranes.
indicating protein cluster formation. The AFM images resolve
STxB clusters on the protein-decorated lo phase with
Dh(lo+STxB_clusters/lo+STxB) ¼ 0.7 ꢂ 0.2 nm (n ¼ 24). In addition,
the overall height of the lo phase as a result of protein binding
increases only by 0.2 nm to Dh(lo+STxB/ld) ¼ 1.0 ꢂ 0.2 nm (n ¼ 69)
indicating low protein density (ESI, Fig. S3†).
Membranes containing Gb3-C24:1 are quite distinct from the
other Gb3-containing membranes as they phase-separate into
three different phases. Upon STxB binding, the overall phase-
separation did not change. Fig. 4A depicts a Bodipy-PC uo-
rescence micrograph again showing three different intensities.
The disordered phase is assigned to the bright green uorescent
phase, the intermediate phase to that with the lower uores-
cence intensity and the dark one to the ordered phase.
Discussion
There are several lines of evidence that membrane bending
leading to the observed Shiga toxin invaginations in cells and
GUVs are preceded by a local compaction of lipids in one leaet
leading to an asymmetric reduction in membrane area.9,11 Such
local lipid compaction is accompanied by protein cluster
formation and lipid sorting.9,40 On the other hand, protein
Fig. 4B shows the localization of Cy3-labeled STxB. Inter-
estingly, the protein only binds to one of the three phases,
binding studies suggest that the binding capacity of
a
Table 2 Ratios of the different membrane phases (lo: liquid-ordered, li: liquid-intermediate) in DOPC/SM/Chol/Gb3 (40 : 35 : 20 : 5) bilayers
doped with different Gb3s on mica and height differences observed in AFM images after STxB binding. The ratios were determined by pixel
analysis using fluorescence images. Height differences were extracted by fitting Gaussians to the height histograms (number of analysed
fluorescence images and height histograms is given in brackets)
Compound lo/%
li/%
Dh(li/ld)/nm
Dh(lo+STxB_clusters/lo+STxB)/nm Dh(lo+STxB_clusters/ld)/nm Dh(lo+STxB/ld)/nm Dh(lo+STxB/li)/nm
27
28
29
30
76 ꢂ 11 (52)
—
—
0.8 ꢂ 0.1 (6)
1.5 ꢂ 0.5 (19)
0.7 ꢂ 0.2 (17)
2.6 ꢂ 0.6 (26)
1.0 ꢂ 0.2 (69)
2.7 ꢂ 0.5 (44)
—
43 ꢂ 7 (64)
77 ꢂ 5 (62)
87 ꢂ 7 (42)
31 ꢂ 8 1.3 ꢂ 0.7 (12)
—
—
1.9 ꢂ 0.6 (15)
—
—
—
—
0.7 ꢂ 0.2 (24)
2.1 ꢂ 0.5 (12)
—
—
—
—
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membrane for STxB depends on the fatty acid of Gb3.15,19,41
The lipid mixture containing Gb3-C24:0-2OH exhibits about
Thus, we asked the question whether protein cluster formation 20% less ordered phase (56% area fraction of lo phase) under
and lipid sorting leading to local lipid compaction is altered by the same experimental conditions before protein addition and
the fatty acid composition of Gb3, which would then have great thus suggests that the lo phase is mainly composed of the
impact on the propensity of a membrane to invaginate as a sphingolipids and cholesterol, which would sum up to a theo-
result of STxB binding. We will rst discuss the individual retical area fraction of about 50%,42 if one takes only the
observations made with the four synthetically derived Gb3 geometry of the lipid molecules into account (ESI†). However,
molecules, which sum up to more than 50% of the Gb3s found aer STxB binding the area percentage of the lo phase increases
in natural mixtures,14,24,25 and then discuss the impact on STxB signicantly to 77%. It has been shown by others that acyl chain
cluster formation and toxin invasion.
hydroxylation only slightly changes the transition temperature
In case of Gb3-C24:0 with a long chain saturated fatty acid, of ternary lipid mixtures but affects the interlipid interaction,
the assigned lo phase amounts to 74% of the total membrane e.g. attenuates the interaction with sterols leading to a uid-
area. Incubation with STxB did not alter this percentage indi- ization of the bilayer.16 This implies that part of the DOPC
cating that no gross lipid reorganization between the two pha- molecules has been relocated from the ld to the lo phase upon
ses takes place. We suggest that the Gb3 molecules are protein binding. In a similar fashion, the area percentage of
preferentially localized in lo domains with the saturated fatty membranes containing Gb3-C24:1-2OH increases by roughly
acid chain interacting with cholesterol resulting in a tightly 20% upon protein binding. Scheve et al.43 reported that, owing
packed lo phase before as well as aer STxB binding (Fig. 6).
to the sterical demand of a protein to bind to its receptor, it
Fig. 6 Schematic drawing of the phase behaviour of lipid membranes induced by different Gb3 species in absence and presence of STxB. For
simplicity, Gb3 molecules have only been drawn in the upper leaflet of the bilayer. The localization of the Gb3s prior to protein binding is only
hypothesized. (27) Membranes doped with Gb3-C24:0 segregate into lo and ld phase with an lo phase area percentage of more than 70%. Binding
of STxB does not lead to lipid redistribution between the phases. After STxB binding Gb3 is located in the lo phase and STxB demixes the lo phase
by clustering the Gb3 leading to STxB clusters. (29) Membranes doped with Gb3-C24:0-2OH exhibit a low lo area percentage due to a presumably
tight lipid packing excluding DOPC. STxB binding recruits and clusters Gb3 in the ordered phase leading to protein clusters. Additionally, the lo
phase area percentage is increased indicating that Gb3 recruitment allows for inclusion of DOPC in the lo phase. (30) Prior to protein binding
membranes containing Gb3-C24:1-2OH show a similar behaviour as described for 27. Interestingly, protein binding again leads to a strong
increase of the lo phase area fraction. The high protein density atop the lo phase indicates steric pressure between proteins bound to the receptor
lipids inducing a mixing of the phases.43 (28) Gb3-C24:1 induces a demixing of the membrane in absence of protein resulting in a third inter-
mediate phase differing in height and fluorophore partition. STxB selectively binds only to one of the three phases showing that Gb3-C24:1 is
solely located in this phase.
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induces an increased lipid mixing eventually leading to the cholesterol enriched phases regardless of the fatty acid attached
disruption of lo/ld phase separation, a phenomenon that could to the molecule.40,50 Membranes containing Gb3 with a satu-
also explain the gross relocalization of DOPC from the lo to the rated fatty acid show densely packed protein clusters sur-
ld phase upon STxB binding.
rounded by less compact bound STxB. In contrast, STxB binds
Unsaturated Gb3 induces a visible demixing of the ordered rather homogeneously and with high density to the lo phase of
phase, which we termed li and lo phase according to their membranes containing Gb3 with an unsaturated fatty acid. This
uorescence intensities in the uorescence images. Just result might be correlated with the nding that the membrane
recently, Sodt et al.44 reported on substructures within the binding capacity for STxB is larger in case of unsaturated
liquid-ordered phase of lipid bilayers as obtained by molecular Gb3s.15,19 Given the affinity constants of STxB to Gb3, all binding
dynamics simulations. These substructures, identied in a 10 sites on the membrane are expected to be occupied.13 However,
ms all-atom trajectory of lo/ld coexistence were composed of the number of Gb3 molecules bound to a STxB pentamer might
saturated hydrocarbon chains packed with local hexagonal vary for the different Gb3-doped membranes. From the crystal
order and separated by interstitial regions enriched in choles- structure of STxB co-crystallized with a soluble receptor
terol and unsaturated chains. In our case, the unsaturated Gb3 analogue, it is known that up to 15 Gb3 molecules can bind to
molecules might foster such demixing of the lo phase interact- the pentamer resulting in dense packing of the receptor
ing preferentially with the unsaturated species of bovine brain lipids.41,51 If a large number of Gb3s is bound to the STxB pen-
sphingomyelin (ESI, Table S2†) and cholesterol and separate tamer, the membrane gets depleted in Gb3 resulting in localized
from the saturated species of bovine brain sphingomyelin.45 protein clusters rather than in a homogeneously covered
Even though the molecules are unsaturated, it is expected that membrane surface. As a lower limiting case, we assumed that all
they still exclude the bulky uorophore, as it has been shown in 15 binding sites of STxB are occupied with Gb3, which would
monolayer experiments that ceramides with an unsaturated result in a theoretical protein surface coverage of roughly 16%
fatty acid tightly pack despite their large steric demand.46 As (ESI†). From the AFM images (Fig. 3C), we estimated a 15%
there is not only a difference in uorophore partition between surface coverage for the STxB clusters on Gb3-C24:0 containing
the two observed phases but also a height difference as obtained membranes, which is in a similar range as the theoretically
by AFM images, we suggest that Gb3-C24:1 is localized only in estimated value. However, as there are also proteins bound to
one of the two phases thus increasing the height owing to the the membrane but not in clusters, one can state that the
size of the Gb3 head group.47 We cannot unambiguously decide number of Gb3 molecules bound per STxB is less than 15 but
whether the li or lo phase in the uorescence micrographs probably very high. Receptor clustering as a result of toxin
contains the Gb3, as the area percentage of the domains are binding was also described for Cholera toxin subunit-B result-
similar but given the percentage of ld phase (24% for Gb3-C24:1 ing in less protein surface coverage at higher GM1 concentra-
compared to 26% for Gb3-C24:0) we assume the two phases to tions.52 In contrast, the unsaturation of the fatty acid in Gb3
be the result of segregation of the lo phase.
increases the area demand of this molecule, which might
Using the phase diagram and tie-lines gathered from uo- decrease the number of Gb3s bound to a pentamer. In this case,
rescence micrographs of GUVs reported by Bezlyepkina et al.,42 we indeed observed a more homogeneous but still high protein
one can estimate the compositions and area fractions of the lo surface coverage on the lo phase. Taking full protein coverage
and ld phases. In general, the area fraction we found for the ld into account would theoretically lead to about 6–10 Gb3-C24:1
phase in our SSMs composed of DOPC/SM/cholesterol (2 : 2 : 1) molecules and about 3–5 Gb3-C24:1-2OH molecules bound per
is lower than that found for GUVs (63%), which probably pentamer (ESI†), which is still a sufficiently large number to
reects the inuence of the solid support (ESI†). Despite this strongly bind the protein onto the membrane taking into
general trend, we found that independent of whether we theo- account that the high affinity binding sites of the protein are
retically replace either 5 mol% of SM or DOPC with 5 mol% of occupied rst.51 Of note, there are indications that Gb3 might be
unsaturated Gb3 in the DOPC/SM/cholesterol (2 : 2 : 1) lipid buried as a result of glycosphingolipid specic interactions with
mixture, this does not explain the observed changes in ld area cholesterol, an aspect that we cannot exclude as all membranes
fraction suggesting that the Gb3 inuences also the contained cholesterol.12,53,54
surrounding lipids (ESI, Fig. S5†). We expect to observe the
Our rough calculations suggest that the number of recruited
same behaviour when using 1-palmitoyl-2-oleoyl-sn-glycero-3- Gb3 molecules to the STxB pentamer is greatly inuenced by
phosphocholine bearing one saturated and one unsaturated the fatty acid of the Gb3 molecule. This difference in lipid
fatty acid chain instead of DOPC as the general phase behaviour recruitment also inuences the membrane organization more
is very similar,37,48 even though POPC can stabilize nanoscopic globally. In case of membranes containing Gb3 with an
domains.49
a-hydroxylated fatty acid, a signicant protein-induced lipid
Aer STxB binding, a change in lateral membrane organi- resorting is observed resulting in a signicantly increased lo
zation as well as protein cluster formation was expected. phase area fraction (21% for Gb3-C24:0-2OH, 17% for Gb3-
However, in contrast to our previous study,9 where we used a C24:1-2OH). However, neither for Gb3-C24:1 nor for Gb3-C24:0
Gb3 mixture, protein cluster formation as well as membrane containing membranes a substantial increase in lo phase area
reorganization strongly depended on the used Gb3 molecule. fraction was observed.
We found that STxB binds to the lo phase demonstrating the
From our results we conclude that the unsaturated Gb3
localization of the different Gb3s with the sphingomyelin/ species are the most important ones for the rst step of Shiga
3112 | Chem. Sci., 2014, 5, 3104–3114
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toxin internalization. Even though they have a much larger area Council (Programme “Ideas” – call identier: ERC-2011-StG
demand than the saturated species, they still get clustered 282105). O.M.S. thanks the GGNB (PBCS) for a Ph.D. fellowship,
underneath the protein. This in turn would lead to an asym- A.R. thanks the Konrad-Adenauer Foundation for a Ph.D.
metric reduction in membrane area, which is proposed to be fellowship. The authors thank Jutta Gerber-Nolte for technical
prerequisite for membrane invaginations. This idea can be assistance.
supported by the observation that in case of Gb3-C24:1 the lo
phase gets reorganized and further phase-separated leading to a
STxB decorated phase that contains more unsaturated lipid
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