Synthesis of Gb3 Glycosphingolipids with Labeled Head Groups: Distribution in Phase-Separated Giant Unilamellar Vesicles

Article abstract of DOI:10.1002/anie.201910148

The receptor lipid Gb₃ is responsible for the specific internalization of Shiga toxin (STx) into cells. The head group of Gb₃ defines the specificity of STx binding, and the backbone with different fatty acids is expected to influence its localization within membranes impacting membrane organization and protein internalization. To investigate this influence, a set of Gb₃ glycosphingolipids labeled with a BODIPY fluorophore attached to the head group was synthesized. C₂₄ fatty acids, saturated, unsaturated, α-hydroxylated derivatives, and a combination thereof, were attached to the sphingosine backbone. The synthetic Gb₃ glycosphingolipids were reconstituted into coexisting liquid-ordered (l_o)/liquid-disordered (l_d) giant unilamellar vesicles (GUVs), and STx binding was verified by fluorescence microscopy. Gb₃ with the C_24:0 fatty acid partitioned mostly in the l_o phase, while the unsaturated C_24:1 fatty acid distributes more into the l_d phase. The α-hydroxylation does not influence its partitioning.

Full text of DOI:10.1002/anie.201910148

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International Edition: DOI: 10.1002/anie.201910148
German Edition: DOI: 10.1002/ange.201910148
Membranes
Synthesis of Gb3 Glycosphingolipids with Labeled Head Groups:
Distribution in Phase-Separated Giant Unilamellar Vesicles
Jeremias Sibold+, Katharina Kettelhoit+, Loan Vuong, Fangyuan Liu, Daniel B. Werz,* and
Claudia Steinem*
Abstract: The receptor lipid Gb3 is responsible for the specific
internalization of Shiga toxin (STx) into cells. The head group
of Gb3 defines the specificity of STx binding, and the backbone
with different fatty acids is expected to influence its localization
within membranes impacting membrane organization and
protein internalization. To investigate this influence, a set of
Gb3 glycosphingolipids labeled with a BODIPY fluorophore
attached to the head group was synthesized. C24 fatty acids,
saturated, unsaturated, a-hydroxylated derivatives, and a com-
bination thereof, were attached to the sphingosine backbone.
The synthetic Gb3 glycosphingolipids were reconstituted into
coexisting liquid-ordered (lo)/liquid-disordered (ld) giant uni-
lamellar vesicles (GUVs), and STx binding was verified by
fluorescence microscopy. Gb3 with the C24:0 fatty acid parti-
tioned mostly in the lo phase, while the unsaturated C24:1 fatty
acid distributes more into the ld phase. The a-hydroxylation
does not influence its partitioning.
called raft domains, which are enriched in sphingomyelin
(SM) and cholesterol (Chol).[2–4] Their size, chemical compo-
sition, and physical characteristics are tightly associated with
their signal processing capabilities.[5] Raft domains are
supposed to have diameters of 10–200 nm and are highly
dynamic structures.[3,6] This combination of smallness and
dynamics bears the major challenge in visualizing raft
domains in cellular membranes.[4] Hence, two approaches
have been pursued within the last decades to shed some light
on the structure and function of these domains. On the one
hand, detergent-resistant membranes were extracted from
cells and their composition analyzed, however they turned out
to be prone to artefacts.[7] On the other hand, artificial
membranes with lipid compositions resembling the outer
leaflet of the plasma membrane were reconstituted, which
separate into a liquid-disordered (ld) and a liquid-ordered (lo)
phase.[8] Typical lipid compositions comprise a low-melting
glycerophospholipid, a high-melting glycerophospholipid or
SM, and Chol.[9] The ld phase has loose lateral lipid packing,
acyl chains with gtg kinks, and fast lateral diffusion. In
contrast, the lo phase is characterized by a tighter lipid
packing and a higher degree of order, but still rather fast
lateral diffusion.[10] However, the size and physical properties
of lo domains formed in artificial membranes are very
different from those found in the plasma membrane. This
difference becomes obvious if comparing, for example, the
physicochemical properties of coexisting lo/ld phase-separated
GUVs with those of phase-separated cell-derived membranes
termed giant plasma membrane vesicles (GPMVs).[11] Despite
this difference between the natural and artificial membrane
systems, artificial coexisting lo/ld membranes have been
frequently used to analyze the partitioning of receptor lipids
and proteins,[12] such as bacterial toxins, in the different
phases.[13]
Introduction
The eukaryotic plasma membrane of animals is a hetero-
geneous structure with a plethora of different lipids. The main
lipid components are glycerophospholipids, sterols, and
sphingolipids.[1] Among them, glycosphingolipids serve a par-
ticular role. They are found in the outer leaflet of the plasma
membrane and are discussed to reside preferentially in so-
[*] J. Sibold,[+] L. Vuong, F. Liu, Prof. Dr. C. Steinem
Georg-August-Universitꢀt Gçttingen, Institute of Organic and Bio-
molecular Chemistry
Tammannstr. 2, 37077 Gçttingen (Germany)
E-mail: claudia.steinem@chemie.uni-goettingen.de
Dr. K. Kettelhoit,[+] Prof. Dr. D. B. Werz
Technische Universitꢀt Braunschweig, Institute of Organic Chemistry
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: d.werz@tu-braunschweig.de
Bacterial toxins are known to bind to specific glycosphin-
golipids embedded in the outer leaflet of the plasma
membrane. Cholera toxin (CTx) produced by Vibrio cholerae
and Shiga toxin (STx) produced by Shigella dysenteriae and by
enterohemorrhagic strains of Escherichia coli, both belonging
Homepage: http://www.werzlab.de
Prof. Dr. C. Steinem
Max Planck Institute for Dynamics and Self Organization
Am Faßberg 17, 37077 Gçttingen (Germany)
to the class of AB5 toxins,[14] bind specifically to monosialote-
[+] These authors contributed equally to this work.
[15]
trahexosylganglioside (GM1
)
and globotriaosyl ceramide
(Gb3),[16,17] respectively. While the head groups of the
glycosphingolipids indeed define the specificity of protein
binding, not much attention has been drawn to the variability
of the ceramide backbone harboring different fatty acids. In
various cell types (human colon Caco-2, HCT-8 epithelial
cells, human endothelial cell lines, primary human umbilical
vein endothelial cells, primary human endothelial cells of the
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201910148.
ꢁ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
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brain and the kidney,[18] and references therein), a conserved
repertoire of Gb3 species was found carrying saturated C16:0
22:0, or C24:0 fatty acids as well as the unsaturated C24:1 fatty
,
C
acid. Results of Lingwood and co-workers[19] suggest that the
pathogenic outcome of Shiga toxin producing E. coli (STEC)
infections is related to the different Gb3 species. To gather
more molecular information, artificial membranes doped with
Gb3 were employed. In coexisting lo/ld supported lipid
membranes, Gb3 species differing in their fatty acid gave rise
to a different phase behavior before and after binding of the B
subunits of STx (STxB) as well as differences in the protein
organization on the membrane surface.[20,21] In giant unila-
mellar vesicles (GUVs), Gb3 species with an unsaturated acyl
chain caused the formation of tubular invaginations upon
STxB binding, in contrast to Gb3 with a saturated acyl
chain.[22] In all these studies, it became evident that STxB
binds exclusively to the lo phase, which also implies that the
receptor Gb3 is localized in the lo phase after protein binding.
However, it remains unclear how Gb3 is distributed in
coexisting lo/ld membranes prior protein binding.
To get access to this information, an approach based on
fluorescently labeled Gb3 molecules can be pursued. How-
ever, it turned out that, if a fluorescent label is attached to the
fatty acid position to ensure that the STxB interaction with
the head group is not influenced by the fluorophore, binding
of STxB is greatly altered.[23] If a fatty acid labeled Gb3 is
reconstituted into lo/ld phase-separated GUVs, the protein
binds to the ld phase and not to the lo phase as known from
membranes containing naturally occurring Gb3.
To date, only a few examples are found in the literature
where synthetic routes towards glycosphingolipids with
labeled head groups have been described.[24] Here, we decided
on a new strategy in line with approaches pursued for GM1 and
GM3[25] and focused on head group labeled Gb3. The idea is to
develop fluorescently labeled Gb3 glycosphingolipids without
altering its binding properties to STxB. We attached a fluo-
rophore via an oligoethylene glycol spacer to the 2’-OH group
of the middle galactose of the Gb3 head group, which is not
involved in STxB binding as deduced from crystal structure
analysis[17] and binding studies of different trisaccarides.[26]
This approach in turn allows us to alter the fatty acid of the
Gb3 molecules.
Scheme 1. Retrosynthetic analysis of head group labeled Gb3PEGnR
derivatives (n=3,13, R=different fatty acids C24:0H, C24:0OH, C24:1
H
and C24:1OH).
a modular convergent synthesis in which a variation of the
fatty acid and the fluorophore is possible with minimal
synthetic effort (Scheme 1). In contrast to semisynthetic
approaches, a convergent total synthesis ensures the highly
defined nature of the obtained material, which was crucial for
our biophysical experiments. The retrosynthetic analysis of
the desired structures 1–8 led to four different components.
The commercially available BODIPY dye 9 should be
attached to the carbohydrate head group in the last step of
the synthesis by a Huisgen cycloaddition (click chemistry).
The sphingosine core should be introduced as the azido
sphingosine 10. The azide serves as a masked amine which
undergoes amide coupling with the four selected fatty acids
(11–14) with a C24 backbone. Assembling the globotriose
building blocks 15 and 16, in which the 2-hydroxy group of the
middle galactose was modified with the PEG linker and the
reducing end was activated for the glycosylation reaction with
10, would be the most challenging endeavor during this
synthesis. Monosaccharide building blocks with carefully
chosen patterns of temporary and permanent protecting
groups had to be synthesized starting from the simple
monosaccharides d-glucose and d-galactose.
Naturally occurring Gb3 molecules carry 24 carbon long
fatty acids, either saturated or monounsaturated.[30] The
galactosyl trichloroacetimidate 17, galactosyl phosphate 18,
and glucoside 19 were identified as suitable precursors to
build up the trisaccharide (Scheme 2). They were prepared
according to literature procedures.[31] The union of 18 and 19
under Lewis-acidic conditions utilizing TMSOTf as a promot-
er afforded the respective (1!4)-linked disaccharide. Perfect
b-selectivity was observed because of the neighboring-group
Results and Discussion
We synthesized a set of Gb3 sphingolipids as depicted in
Scheme 1. Altogether eight different glycosphingolipids were
synthesized and they consist of the globotriaose head group
with two different oligoethylene glycol (PEG) linkers, to
which a BODIPY fluorophore was attached and the sphin-
gosine. Saturated, unsaturated, a-hydroxylated derivatives,
and a combination thereof were prepared, all based on a C24
fatty acid. C24 fatty acids were chosen as they are the major
constituent (> 50%) found in natural Gb3 mixtures such as
toxin insensitive erythrocytes,[27] HeLa-cells,[28] and HEp-2
cells.[29]
To access the head group labeled Gb3 derivatives with
different fatty acids and PEG linker lengths we designed
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part of the glycosphingolipid.[21] To attach the PEG linker, the
double bond of the pentenyl handle was first transformed into
a thioester with tioacetic acid under radical conditions. This
species was hydrolyzed under basic conditions and the
emerging highly nucleophilic thiol was subsequently reacted
with the PEG bromides 21 (13 ethylene glycol units) and 22 (3
ethylene glycol units). To ensure a full protection of all
hydroxy groups, the benzoylation step was repeated. Finally,
the anomeric protecting group was removed with trifluoro-
acetic acid and the reducing end was converted into the
corresponding trichloroacetimidates 15 and 16.
To build up the glycolipid, the trichloroacetimidates were
reacted with the protected azidosphingosine 10,[33] which was
synthesized starting from the chiral pool compound l-serine
(for detailed information see the Supporting Information), in
a glycosylation reaction utilizing TMSOTf as the Lewis acid
to afford 23 and 24 in moderate yields (Scheme 4). Comparing
experiments with globotriaosyl trichloroacetimidates devoid
of the PEG modification indicated that the Lewis-basic linker
might hamper this very sensitive glycosylation step. Stau-
dinger reduction of the azides and direct coupling with the
fatty acids 11–14,[21,34] without isolating the intermediary
amines, afforded the PEG-modified glycosphingolipids 25–32.
Global deprotection under Zemplꢀn conditions set the stage
Scheme 2. Assembly of the Gb3 trisaccharide.
participation of the Fmoc group of 18. During the course of
the reaction the para-methoxybenzyl group of the galactose
was cleaved,[32] yielding the lactose acceptor for the second
glycosylation step with 17 under Lewis-acidic conditions
without any additional deprotection step. The desired a-
configured product was isolated as the main product when
diethyl ether was used as a cosolvent. Subsequent removal of
the Fmoc protecting group with piperidine led to the
trisaccharide 20.
The trisaccharide 20 was then equipped with a pentenyl
chain in the position where the fluorophore needs to be
attached (Scheme 3). In the next step the substrate was
subjected to Birch conditions to remove all benzyl protecting
groups. Despite the strongly reducing conditions, the anome-
ric CH2CH2TMS group and the pentenyl handle stayed intact.
Deprotection was followed by DMAP-mediated benzoyla-
tion. In contrast to benzyl groups, benzoyl esters have the
advantage that they can be easily removed at the end of the
synthetic route without affecting the double bond in the lipid
Scheme 3. Synthesis of the trichloroacetimidates 15 and 16 with two
different PEG linkers.
Scheme 4. Gb3 glycosphingolipid assembly.
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for the final step of the synthesis. The commercially available
BODIPY dye 9 was introduced into the glycosphingolipids by
coupling its azide unit with the alkyne moiety of the PEG
linker under mild copper(I)-catalyzed conditions (Scheme 5).
Figure 1. Confocal images of a phase-separated GUV composed of
DOPC/SM-porc/Chol/1/Texas Red-DHPE (39.75/35/20/5/0.25) in an
aqueous solution of STxB-Cy5 (500 nm, monomer). A) Texas Red-DHPE
fluorescence (red). B) STxB-Cy5 fluorescence (green). The yellow lines
indicate the position of the fluorescence intensity profiles shown below
the images.
To quantitatively compare the phase partitioning among
the different Gb3 species, we used the fluorophore Dy731-
DOPE as ld marker[23] to guarantee that the fluorescence of
the BODIPY labeled Gb3 does not spectrally overlap with the
absorption of the ld marker. Moreover, the concentration of
Gb3 was reduced from 5 to 1 mol% to ensure that self-
quenching of the BODIPY fluorophore is minimized (see
Figure S1 in the Supporting Information). GUVs composed
of DOPC/SM-porc/Chol/Gb3/Dy731 (39/39/20/1/1) were pre-
pared. As it is known that the composition of GUVs obtained
by electroformation is rather heterogeneous,[35] at least two
independent GUV preparations with about 30 individual
GUVs each were analyzed. Confocal z-stack images were
measured for each GUV and line profiles were taken from
each slice, where phase separation was visible. An example of
fluorescence images of a lo/ld coexisting GUV together with
the line profile is shown in Figure 2. The fluorophore Dy731-
DOPE indicates the ld phase (Figure 2A).[23] From the
BODIPY fluorescence intensity (Figure 2B), the preferential
localization of 1 is visible. To quantify the partition of 1, the
BODIPY intensity of the ld phase (I(ld)) and of the lo phase
(I(lo)) as obtained from the corresponding line profile was
determined and the lo distribution (%lo) was calculated
[Eq. (1)]:[23]
Scheme 5. Huisgen cycloaddition of the BODIPY derivative 9 with the
glycosphingolipids 25–32.
In total, eight different fluorescently labeled glycosphingoli-
pids (1–8), varying in the PEG linker length and the acyl chain
of the fatty acid, were obtained. The linker length (n) is either
3 or 13 oligoethylene glycol groups. The fatty acid (Cm:D) is
either saturated (C24:0) or unsaturated (C24:1). Hydroxylation
at the a-position is indicated by OH, and non-hydroxylation is
indicated by H.
Starting with the saturated C24 fatty acid and a PEG
spacer composed of 13 oligoethylene glycol units, we pre-
pared GUVs composed of the well-known raft mixture 1,2-
dioleoyl-sn-glycero-3-phosphocholine
(DOPC)/SM-porc/
Chol labeled with 5 mol% 1 and 0.25 mol% Texas Red-
DHPE (39.75/35/20/5/0.25) to address the question of whether
STxB is indeed still capable of binding to the head group
modified Gb3 and whether it binds to the lo phase as expected.
Figure 1 shows representative confocal images of a GUV
in a 500 nm STxB-Cy5 (monomer) solution. Texas Red-
DHPE partitions preferentially in the ld phase, visualizing the
coexisting lo/ld membrane (Figure 1A). The fluorescence
image of STxB-Cy5 shows that STxB binds to the GUV and
that it binds to the lo phase (Figure 1B). This result confirms
our hypothesis that the fluorescent label at the 2’-OH position
does not greatly interfere with the binding properties of STxB
and is suited to investigate the partition of different Gb3
species as a function of the fatty acid in coexisting lo/ld
membranes.
IðloÞ
ð1Þ
%lo ¼
IðloÞ þ IðldÞ
Several tens of line profiles were taken from each GUV.
All %lo values were cast into a histogram (Figure 2C). Data
obtained in this manner are presented as violin plots
throughout the manuscript.
There is increasing evidence that the size of the linker
attached to the head group of a lipid alters the phase behavior
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Figure 3. lo distribution of different Gb3 species in GUVs composed of
DOPC/SM-porc/Chol/Gb3/Dy731 (39/39/20/1/1). The partition of Gb3
sphingolipids with the short PEG linker (PEG3) was compared with
those carrying the long PEG linker (PEG13). The mean values are given
as a red star, while the red solid lines show the median value
(Table 1).
Figure 2. Confocal images of a phase-separated GUV composed of
DOPC/SM-porc/Chol/1/Dy731-DOPE (39/39/20/1/1). A) Dy731-DOPE
fluorescence (red). B) 1 fluorescence (green). The yellow lines indicate
where the fluorescence intensity profiles (bottom images) were ob-
tained. From the intensity profiles %lo =68.2% was calculated for 1.
C) Histogram and corresponding violin plot obtained from 60 GUVs
(number of line profiles atop) with the composition as in (A/B). The
red solid line indicates the median value, the red star the mean value.
Table 1: Mean values of the lo distributions (%lo) for the different Gb3
sphingolipids in GUVs composed of DOPC/SM-porc/Chol/Gb3/Dy731
(39/39/20/1/1).
No.
Gb3
%lo (N)
1
2
3
4
5
6
7
8
Gb3PEG13C24:0
H
0.74ꢀ0.07 (2525)
0.41ꢀ0.07 (2516)
0.71ꢀ0.05 (3064)
0.42ꢀ0.08 (2273)
0.47ꢀ0.15 (1654)
0.32ꢀ0.07 (2351)
0.50ꢀ0.08 (2377)
0.27ꢀ0.06 (2701)
Gb3PEG3C24:0
H
Gb3PEG13C24:0OH
Gb3PEG3C24:0OH
of the fluorescently labeled lipid.[36,37] To investigate whether
the linker length, that is, the number of ethylene glycol units,
influences the partition of the Gb3 sphingolipids in phase-
separated GUVs, we synthesized Gb3 molecules differing in
their fatty acid with either 13 ethylene glycol units (PEG13) or
3 (PEG3). Independent of the fatty acid, the same trend is
observed (Figure 3). All Gb3 sphingolipids with PEG13
partition more in the lo phase than the corresponding Gb3
species with PEG3.
Gb3PEG13C24:1
H
Gb3PEG3C24:1
H
Gb3PEG13C24:1OH
Gb3PEG3C24:1OH
The errors are the standard deviation of the mean. N=number of line
profiles.
Table 2: Differences in the mean values dependent on the functional
group.
The mean values are summarized in Table 1. The differ-
ence between the lo distribution of PEG13Gb3 species and
PEG3Gb3 species lies between 0.15 and 0.33 (Table 2, DPEG).
Such altered partitioning of a lipid as a function of linker
length, to which a fluorophore has been attached, was also
observed by Honigmann et al.[36] They reported on a fluoro-
phore that was either directly connected to the lipid 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or
connected by a PEG-linker with 45 ethylene glycol units,
and was reconstituted into supported lipid membranes
composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine
(DPhPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC)/Chol. A fluorescence analysis of the partition clearly
showed that the fluorescent lipid lacking the PEG-linker was
preferentially localized in the ld phase, while that with the
PEG-linker partitioned into the lo phase. Similarly, Momin
et al.[38] and Bordovsky et al.[37] found that an increase in
DPEG
DC24
DOH
1-2:
3-4:
5-6:
7-8:
0.33ꢀ0.14
1-5:
3-7:
2-6:
4-8:
0.27ꢀ0.22
1-3:
5-7:
2-4:
6-8:
0.03ꢀ0.12
0.29ꢀ0.13
0.15ꢀ0.22
0.23ꢀ0.14
0.21ꢀ0.13
0.09ꢀ0.14
0.15ꢀ0.14
0.03ꢀ0.23
ꢁ0.01ꢀ0.15
0.05ꢀ0.13
DPEG=%lo (PEG13)—%lo (PEG3); DC24=%lo (C24:0)—%lo (C24:1);
DOH=%lo (H)—%lo (OH).
length of the hydrophilic PEG linker at the head group of
lipids that are expected to be localized in the lo phase of
coexisting lo/ld membranes is required to favor their parti-
tioning in the lo phase. This observation is explained by the
notion that the fluorophore itself is partially hydrophobic and
might be also bulky. It changes the packing parameter of the
lipid. If the fluorophore is directly connected to the lipid or
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attached by a short linker, the size of the lipidꢁs head group is
either the sphingolipid or mixed with palmitoyl sphingomye-
expanded and the lipid is more conically shaped, favoring the
ld phase.[39] For the slightly hydrophobic but small BODIPY
fluorophore used in our study, a hydrophilic PEG spacer of
suitable length is required to mitigate interactions with the
membrane. Momin et al.[38] found a linker with 10 ethylene
glycol units to be sufficient to decouple the fluorophore from
the membrane.[39] In our study, a 13-unit long linker decou-
pled the fluorophore from the membrane interface with the
result that 1, which is expected to at least preferentially
partition into the lo phase, indeed has a lo distribution of
almost 0.75. From these results, we conclude that the Gb3
species with PEG13 are better suited to report on the natural
partition of Gb3 than those with PEG3. Thus, the experiments
in which we compare the influence of unsaturation and
hydroxylation of the fatty acid of Gb3 are all performed with
the PEG13 species. The corresponding results with the PEG3
linker can be found in the Supporting Information (see
Figures S2 and S3).
lin.[40] This behavior was also found by Mate et al.,[42] who
reported that sphingomyelin with the C24:0 fatty acid recon-
stituted into a DOPC/Chol membrane leads to visible phase
separation into an lo and ld phase, while the sphingomyelin
with the C24:1 fatty results only in one lipid phase.
These results support our notion that the packing of the
unsaturated Gb3 species disfavors its partition in the lo phase.
Similar to our in vitro results, Legros et al.[43] found in primary
human blood brain barrier endothelial cells that Gb3 with C24:1
fatty acids resides more strongly in non-detergent-resistant
membranes compared to Gb3 with C24:0 fatty acids.
In nature, about 50% of the Gb3 sphingolipids are
decorated with an OH group in the a-position of the fatty
acid, raising the question, whether this OH group alters the
Gb3 partition. The results (Figure 5) clearly indicate that the
OH group in the a-position does not influence its distribution.
The differences of %lo for 1/3 and 5/7 are in the range of
ꢁ0.03–0.03 and are not significant (Table 2, DOH).
We investigated the influence of the fatty acid saturation
on the partition behavior of Gb3 (Figure 4). The results show
that introducing a fatty acid with a cis-double bond redis-
tributes the Gb3 sphingolipid in the ld phase, and can be
rationalized by the increased space requirement of the Gb3
species with the C24:1 fatty acid. The differences between the lo
distribution of 1/5 and 3/7 harboring the PEG13 linker are
significant and range between 0.21 and 0.27 (Table 2, DC24).
Figure 5. lo distribution of different Gb3 species with a PEG13 linker in
GUVs composed of DOPC/SM-porc/Chol/Gb3/Dy731 (39/39/20/1/1).
The partition of the Gb3 sphingolipids, which are nonhydroxylated in
the a-position (H) is compared with that carrying an a-hydroxylation
(OH). The mean values are given as red stars, while the red solid lines
are the median values (Table 1).
Monolayer experiments on galactosyl ceramide (GalCer),
harboring either an a-hydroxylated or nonhydroxylated C24:0
fatty acid on a Langmuir trough, suggest that the a-
hydroxylation does not change the area per lipid at
30 mNmꢁ1,[44] a surface pressure that reflects the packing
Figure 4. lo distribution of different Gb3 species with a PEG13 linker in
GUVs composed of DOPC/SM-porc/Chol/Gb3/Dy731 (39/39/20/1/1).
The partition of the Gb3 species harboring a saturated fatty acid (C24:0
were compared with that with an attached unsaturated fatty acid
(C24:1). The mean values are given as a red star, while the red lines
show the median value (Table 1).
)
2
density of bilayers.[45] Using H NMR spectroscopy, Morrow
and co-workers[41,46] also demonstrated that the order param-
eter of the fatty acids of GalCer embedded in a POPC/Chol
membrane and the orientation of the head group does not
change considerably.
Bjçrkqvist et al.[40] investigated different glycosphingoli-
pids as well as sphingomyelins and found by differential
scanning calorimetry (DSC) that the phase-transition temper-
ature was decreased by about 20 K for all sphingolipids
harboring the C24:1 fatty acid compared to the corresponding
C24:0 sphingolipids, demonstrating their different packing
behavior. The ability to pack tightly with ordered acyl chains
in case of a saturated fatty acid[41] is a requirement for
membrane lipids to partition into lo domains and they
concluded that the C24:1 sphingolipids are less likely to
partition into the lo phase. Fluorescence quenching experi-
ments revealed that sphingolipids with a C24:0 fatty acid form
lo domains in multicomponent membranes composed of
This report is in line with our observation that the OH
group does not significantly alter the partitioning of the Gb3
species in phase-separated GUVs. However, in a previous
study, we found that the 2-OH group influences the fraction of
lo phase in phase-separated supported lipid bilayers.[21] In the
case of the hydroxylated C24:0 fatty acid, the lo fraction was
smaller than that of the nonhydroxylated species. Slotte and
co-workers[47] showed that the 2-OH group increases the
hydration in the membrane interface and decreases the
affinity of a sphingolipid for sterols. The same was found by
Lingwood et al.[48] and Yahi et al.[49] and implies that the
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Figure 6. lo distribution of different Gb3 species with a PEG13 linker composed of DOPC/SM/Chol/Gb3/Dy731 (39/39/20/1/1). The partition of
four different Gb3 sphingolipids in lo/ld phase-separated GUVs with different sphingomyelin species are shown: SM C16:0 (palmitoyl SM), SM C18:0
(stearoyl SM), SM C20:0 (arachidoyl SM), SM C22:0 (behenoyl SM) and SM C24:0 (lignoceroyl SM). The mean values are given as red stars and the
solid red lines represent the median value(Table 3).
Table 3: Mean values of the lo distributions (%lo) for the different Gb3 sphingolipids with the PEG13 linker in GUVs composed of DOPC/SM/Chol/Gb3/
Dy731 (39/39/20/1/1) varying in the SM species.
Gb3
%lo(SM C16:0) (N)
%lo(SM C18:0) (N)
%lo(SM C20:0) (N)
%lo(SM C22:0) (N)
%lo(SM C24:0) (N)
1
3
5
7
0.41ꢀ0.11 (2392)
0.45ꢀ0.10 (3232)
0.24ꢀ0.10 (2035)
0.35ꢀ0.06 (1814)
0.55ꢀ0.17 (2986)
0.59ꢀ0.14 (3396)
0.28ꢀ0.11 (1845)
0.34ꢀ0.08 (2465)
0.74ꢀ0.05 (2397)
0.71ꢀ0.05 (2414)
0.54ꢀ0.11 (2482)
0.46ꢀ0.08 (2648)
0.74ꢀ0.09 (2077)
0.72ꢀ0.07 (2893)
0.45ꢀ0.15 (2759)
0.51ꢀ0.09 (2266)
0.75ꢀ0.09 (1707)
0.77ꢀ0.07 (2509)
0.57ꢀ0.12 (1730)
0.50ꢀ0.10 (2491)
SM C16:0 (palmitoyl SM), SM C18:0 (stearoyl SM), SM C20:0 (arachidoyl SM), SM C22:0 (behenoyl SM), and SM C24:0 (lignoceroyl SM). The errors are the
standard deviation of the mean. N=number of line profiles.
recruitment of Chol into the lo phase by hydroxylated Gb3 is
reduced compared to the nonhydroxylated species, leading to
a smaller lo fraction, while the amount of Gb3 in the lo fraction
is the same.
was—for fatty acids with a length of more than 20 carbon
atoms—not only observed in the gel phase but also in the
liquid-crystalline phase.[52,53] Interdigitation was also reported
for glycosphingolipids carrying a C24 fatty acid.[53,54] Hence, it
is likely that the Gb3 species under investigation preferen-
tially partition into the lo phase if SM interdigitates. Inter-
digitation of SM in the liquid-crystalline phase occurs for C20
fatty acids and longer, in agreement with our observation that
the partition in the lo phase is increased for SM species with
C20 fatty acids or longer.
Our results clearly demonstrate that the fatty acid of Gb3
influences its partitioning into the lo phase. One reason might
be found in the interaction of the Gb3 fatty acid with the fatty
acid of SM, which we next analyzed. To investigate this aspect
in more detail, we replaced the SM mixture isolated from pigs
with synthetic pure SM. Exchanging a sphingomyelin mixture
with sphingomyelins with a defined fatty acid is known to
alter the phase separation behavior of ternary mixtures.[50]
Five different SM species with a saturated fatty acid of
varying length were chosen, namely palmitoyl SM (C16:0),
stearoyl SM (C18:0), arachidoyl SM (C20:0), behenoyl SM
(C22:0), and lignoceroyl SM (C24:0), and the lo distribution of
each Gb3 species in these membranes was determined (Fig-
ure 6, Table 3).
However, the lo phase consists not only of SM but also of
Chol owing to its better solubility in SM membranes than in
[56,57]
PC membranes.[55–57] Chol is best soluble in SM C16:0
.
If
the solubility of Chol in the lo phase greatly influenced the
Gb3 distribution in the lo phase, the opposite trend would have
been observed. This trend was not found and agrees with the
idea that the interaction of Gb3 with Chol is less important
than the one with SM.
The fatty-acid chain length also determines the length
difference between the two hydrophobic chains, which
increases with an increase in fatty-acid chain length. This
mismatch results in interdigitation of both leaflets,[51] which
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Conclusion
GM1 and Gb3 detection by fluorescently labeled Cholera
toxin B subunits (CTxB) and Shiga toxin B subunits (STxB),
respectively is a well-established tool for monitoring lo
membrane domains[58] and implies that these glycosphingoli-
pids are localized in the lo phase. However, as each CTxB and
STxB pentamer can recruit a maximum of 5 (CTx) or 15
(STx) receptor lipids, the glycosphingolipid partitioning in
coexisting lo/ld membranes after protein binding does not
necessarily reflect the situation prior protein binding. Hence,
to be able to quantify the partitioning of Gb3 in phase
coexisting lo/ld membranes by means of fluorescence readout,
chemical access to fluorescently labeled pure Gb3 molecules is
required. The approach of synthesizing head group labeled
glycosphingolipids enables one to address the question how
the fatty acid of a glycosphingolipid influences its distribution
in lo/ld phase-separated membranes, a question that has been
hardly addressed because most of the glycosphingolipids are
not available in chemically pure form. Our results clearly
demonstrate that the fatty acid (un)saturation significantly
shifts the Gb3 molecules from the lo phase (C24:0) to the ld
phase (C24:1). As STxB exclusively binds to Gb3 in the lo phase,
the amount of redistributed Gb3 and probably also other lo
phase lipids thus depends on the fatty acid of Gb3. However,
the a-hydroxylation does not alter the partition of Gb3, even
though it has been shown that the OH group of Chol can form
a hydrogen bond only to the nonhydroxylated fatty acid.
Instead, the length match of the fatty acids of SM and Gb3
appear to play a more decisive role in determining where the
Gb3 glycosphingolipids are preferentially localized. As the
combination of the attached fatty acids of SM and Gb3
considerably impacts the distribution of the Gb3 glycosphin-
golipids, it is conceivable that the overall recruitment of lipids
and thus the Shiga toxin induced membrane reorganization
that eventually leads to the invagination of the protein into
the host cell, is strongly influenced by the fatty acid
composition of Gb3.
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Keywords: carbohydrates · fatty acids · fluorescence ·
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Manuscript received: August 9, 2019
Revised manuscript received: September 9, 2019
Accepted manuscript online: September 17, 2019
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Version of record online: && &&
,
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Research Articles
Membranes
J. Sibold, K. Kettelhoit, L. Vuong, F. Liu,
D. B. Werz,* C. Steinem*
&&&& — &&&&
Synthesis of Gb3 Glycosphingolipids with
Labeled Head Groups: Distribution in
Phase-Separated Giant Unilamellar
Vesicles
Making the invisible visible: Shiga toxin B
subunits (STxB) bind to liquid-ordered
(lo) domains in model membranes,
implying that its receptor Gb3 is localized
in these lo domains. The synthetic access
to Gb3 glycosphingolipids with labeled
head groups allows quantification of their
partitioning in coexisting lo/ld (liquid-dis-
ordered) phase giant unilamellar vesicles
prior to STxB binding. The data clearly
indicate an impact of the fatty acid
(un)saturation and a-hydroxylation on
the partitioning between phases.
Angew. Chem. Int. Ed. 2019, 58, 2 – 11
ꢀ 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
&&&&
These are not the final page numbers!