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Originally published In Press as doi:10.1074/jbc.M201305200 on March 21, 2002
J. Biol. Chem., Vol. 277, Issue 22, 19476-19481, May 31, 2002
Sphingomyelin Modulates the Transbilayer Distribution of
Galactosylceramide in Phospholipid Membranes*,
Peter
Mattjus §,
Barbara
Malewicz§,
Jacob T.
Valiyaveettil§¶,
Wolfgang J.
Baumann,
Robert
Bittman¶, and
Rhoderick E.
Brown
From the University of Minnesota, Hormel Institute,
Austin, Minnesota 55912 and the ¶ Department of Chemistry and
Biochemistry, Queens College of the City University of New York,
Flushing, New York 11367-1597
Received for publication, February 7, 2002, and in revised form, March 20, 2002
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ABSTRACT |
The interrelationships among sphingolipid
structure, membrane curvature, and glycosphingolipid transmembrane
distribution remain poorly defined despite the emerging importance of
sphingolipids in curved regions and vesicle buds of biomembranes. Here,
we describe a novel approach to investigate the transmembrane
distribution of galactosylceramide in phospholipid small unilamellar
vesicles by 13C NMR spectroscopy. Quantitation of the
transbilayer distribution of [6-13C]galactosylceramide
(99.8% isotopic enrichment) was achieved by exposure of vesicles to
the paramagnetic ion, Mn2+. The data show that
[6-13C]galactosylceramide prefers (70%) the inner
leaflet of phosphatidylcholine vesicles. Increasing the sphingomyelin
content of the 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles
shifted galactosylceramide from the inner to the outer leaflet. The
amount of galactosylceramide localized in the inner leaflet decreased
from 70% in pure 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles to
only 40% in 1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin (1:2) vesicles. The present study demonstrates that sphingomyelin can
dramatically alter the transbilayer distribution of a
monohexosylceramide, such as galactosylceramide, in
1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin vesicles. The
results suggest that sphingolipid-sphingolipid interactions that
occur even in the absence of cholesterol play a role in
controlling the transmembrane distributions of cerebrosides.
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INTRODUCTION |
Sphingolipids participate in a number of important cellular
processes that require membrane budding, fission, or vesiculation (1,
2). Examples include infectious processes involving bacterial toxin and
envelope virus entry into cells (3, 4), exosomal antigen presentation
(5), and processes related to the terminal stages of apoptosis (6).
Many recent investigations, by this laboratory and others, have focused
on the in-plane lateral interactions among sphingolipids, cholesterol,
and other membrane lipids (7-10). As a result, significant new
insights into sphingolipid organization in membranes have emerged,
including the identification and characterization of
sphingolipid-enriched, liquid-ordered microdomains, often referred to
as rafts (11-13). With so much emphasis on lipid lateral interactions,
studies of sphingolipid transmembrane distribution have been relatively
few (14), and the interrelationships among sphingolipid structure,
membrane curvature, and glycosphingolipid transmembrane distribution
remain poorly understood (15).
Much of what is currently known about the mechanical forces affecting
membrane curvature has been achieved by investigations of
phosphoglyceride model membranes (16). The elastic constants associated
with a fluid membrane are the bending elastic modulus and the
spontaneous curvature (17, 18). The bending elastic modulus is the
resistance of membranes to curvature or the bending rigidity, whereas
the spontaneous curvature is the inherent curvature of an unconstrained
membrane section and changes with lipid structure. Because biomembranes
are largely bilayers, each leaflet contributes to the overall stiffness
in nonlocalized ways that arise from the different strains to which
molecules in each leaflet are subjected as the bilayer bends. An
outward curvature results in expansion of the outer leaflet of the
bilayer along with a compression of the inner leaflet. The different
strains in each leaflet produce mechanical stress gradients within the
membrane. The stress gradients can significantly increase lateral
diffusivity (19, 20) and be a driving force for the transbilayer
migration of lipid molecules between leaflets (20, 21).
The altered lipid packing and stress gradients in highly curved
membranes can be relieved by the generation of asymmetries in the lipid
transbilayer distributions that depend on the overall molecular shape
of different individual lipids (22). For instance, a well characterized
lipid mass imbalance (2:1) exists in the outer and inner leaflets of
phosphatidylcholine (PC)1
small unilamellar vesicles (SUVs) as a consequence of packing the
roughly cylindrically shaped PC amphiphiles into a highly curved
bilayer vesicle (23, 24). The resulting transbilayer lipid mass
imbalance can be maintained almost indefinitely by keeping the SUVs in
the liquid-crystalline phase state to minimize transient packing
defects that promote slow relaxation processes (25, 26). In SUVs
composed of equimolar egg phosphatidylethanolamine (PE) and egg PC, PE
is enriched in the inner leaflet, whereas the PC is enriched in the
outer leaflet. The smaller and less hydrated headgroup of PE imparts a
cone-like molecular shape which is better suited than PC's cylindrical
shape for inner leaflet localization in highly curved bilayers (27).
Geometric accommodation of lipid shape also has provided a similar,
logical explanation for the transbilayer distributions of lyso-PC/PC
mixtures (28). However, lipid geometric shapes alone do not
satisfactorily account for the transbilayer distributions observed when
PC SUVs contain low mole fractions of either PE or phosphatidylglycerol
(PG). In this case, disproportionately higher amounts of PE or PG are observed in the SUV outer leaflet, putatively because of generalized lattice packing effects (29, 30). Together, these studies show that
investigating lipid transbilayer distributions in vesicles provides an
effective means to gain insights into the interrelationship between
lipid structure and membrane curvature.
The present study was motivated by the need to better understand and
define: 1) the transbilayer distribution of simple sphingolipids in
phospholipid membranes and 2) the impact of changing vesicle composition on sphingolipid transmembrane distribution. Here, we
describe a novel means to quantify the transbilayer distribution of
[13C]galactosylceramide (GalCer) by 13C NMR.
Interestingly, we find that [13C]GalCer preferentially
localizes to the inner leaflets of POPC SUVs. In response to increasing
sphingomyelin (SPM) content, the GalCer transmembrane distribution
shifts markedly toward the outer SUV leaflet even though PC and SPM
have chemically identical polar headgroups. The results suggest that
SPM-GalCer interactions, even in the absence of cholesterol, play an
important role in controlling cerebroside transbilayer distributions.
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EXPERIMENTAL PROCEDURES |
Materials--
POPC and egg SPM were obtained from Avanti Polar
Lipids (Alabaster, AL); bovine brain GalCer without hydroxy fatty acyl
chains, was from Sigma-Aldrich; and
D-erythro-sphingosine, was from Matreya (State
College, PA). [6-13C]Galactose (99 atom % 13C) was obtained from Omicron Biochemicals (South Bend,
IN) and used to synthesize [6-13C]GalCer (99.8% isotopic
enrichment, Fig. 1A). A complete description and a scheme of
the [6-13C]GalCer synthesis are provided in the
Supporting Information.2
Phospholipid concentration was determined by the Bartlett method (31),
and GalCer concentration was quantitated gravimetrically. Deuterated
solvents (CDCl3, CD3OD, D2O) were
obtained from Cambridge Isotope Laboratories (Andover, MA).
Vesicle Preparation--
SUVs were prepared by sonication using
a modification of the established procedure by Huang and Thompson (25).
The total amount of lipid in each preparation was kept constant (200 µmol). The lipids were dissolved in 15 ml of
CHCl3:CH3OH (2:1) in a 50-ml round-bottom
flask. For preparations containing GalCer, a drop of water was added to
aid solubilization. A lipid film was obtained by slowly evaporating the
solvents at 37 °C on a rotary evaporator, followed by freeze-drying
in vacuo for 6 h. The lipid film was hydrated in 2 ml
of D2O, then dispersed by vortexing with intermittent warming to 37 °C, and the dispersion was sonicated under nitrogen for 30-60 min until translucent. After removal of titanium debris by
centrifugation at 50,000 × g for 60 min, the vesicles
were used immediately for NMR analysis. Vesicle stability and vesicle impermeability to ions was ascertained by 31P NMR by
monitoring phospholipid chemical shifts and signal intensities as a
function of time. By these criteria, all vesicles used in the present
study remained stable and ion-impermeable for several days.
Localization of Phospholipids in SUVs by 31P
NMR--
POPC and SPM were localized and quantified in the inner and
outer vesicle leaflets of SUVs by 31P NMR using 1 mM praseodymium (Pr3+) ions as the paramagnetic
shift reagent (28). Proton-decoupled 31P NMR spectra were
recorded at 121.42 MHz on a Varian UNITY 300 instrument (Varian Assoc.,
Palo Alto, CA) using a 5-mm variable temperature probe (37.0 ± 0.1 °C). Standard single-pulse experiments entailed a 90° pulse of
15 µs, an acquisition time of 1.6 s, and a pulse delay of 2 s, with the decoupler gated on during acquisition only. At a
spectral width of 10,000 Hz, 32,000 data points were collected, whereas
1,600 and 6,400 transients were used for samples obtained in the
absence and presence of Pr3+, respectively. Data were then
zero-filled and Fourier-transformed after applying 0.1-Hz exponential
line broadening. Peak areas were digitally integrated. Spectra were
referenced relative to the external standard, concentrated
H3PO4, having a chemical shift ( ) of 0.00 ppm.
Localization of GalCer in Phospholipid SUVs by 13C
NMR--
GalCer was localized and quantified in the inner and outer
leaflets of POPC and POPC/SPM vesicles by 13C NMR using 5 mM Mn2+ as quenching agent. Proton-decoupled
13C NMR spectra of [6-13C]GalCer containing
SUVs were acquired at 75.423 MHz in the absolute mode at 37 °C.
Standard single-pulse measurements entailed a 90° pulse of 9 µs, a
pulse delay of 1 s, and an acquisition time of 1.8 s. At a
spectral width of 16,500 Hz, 59,900 data points were collected, and
24,000 transients were used. Data were zero-filled and
Fourier-transformed after applying 1-Hz exponential line broadening. Peak areas were digitally integrated. The integral of the resonance at
61.361 represented the total [6-13C]GalCer in the
vesicles (see "Results" for details). To quantify the GalCer
localized in the SUV inner leaflet, 5 mM Mn2+
was added to quench the [6-13C]GalCer resonance
associated with the outer bilayer leaflet. The difference between the
integrals of the [6-13C]GalCer resonances observed at 61.361 in the absence (total GalCer) and in the presence (inner GalCer)
of Mn2+ ions provided quantitation of the ion-accessible
GalCer in the outer vesicle leaflet.
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RESULTS |
Novel Approach to Measure Glycolipid Transbilayer
Distribution in Phospholipid Vesicles by 13C
NMR--
Phospholipid transbilayer distribution between the inner and
outer leaflets of vesicles can be accurately determined by
31P NMR (32-34). However, this approach is not suitable
for monitoring the transbilayer distribution of glycosphingolipids
because of the lack of phosphate in the headgroup of these lipids.
Thus, the localization of the sugar headgroups of glycosphingolipids incorporated into vesicles was analyzed by 13C NMR spectroscopy.
A comparison of 13C NMR spectra of phospholipids (35) and
of bovine brain GalCer in solution
(CDCl3:CD3OD:D2O; 50:50:15, v/v/v; Fig. 1B), indicated that two
signature resonances derived from C-6 and C-1 of galactose ( 61.445 and 104.083, respectively) did not overlap with any of the
phospholipid resonances and thus might be used for quantitative
analysis. However, preliminary experiments with vesicles composed of
PC, SPM, and GalCer (40:40:20 mol %) indicated that only the
61.361-ppm resonance derived from C-6 of galactose could be clearly
detected, whereas the C-1 resonance was broadened almost beyond
recognition (data not shown). This finding is consistent with C-1 being
more motionally restricted by virtue of being part of the pyranose ring
and buried in the membrane interfacial region. In contrast, the C-6
carbon is not part of the rigid ring system, can rotate more freely,
and projects farther into the aqueous phase (36). Thus, the
13C NMR resonance of the galactose C-6 carbon was deemed
best suited for quantifying the transbilayer distribution of GalCer in
vesicles.
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Fig. 1.
Solution spectra of GalCer.
A, 13C NMR of [6-13C]GalCer.
B, natural abundance 13C NMR of bovine brain
GalCer (without hydroxy fatty acyl chains). Both spectra were acquired
in CDCl3:CD3OD:D2O (50:50:15
v/v/v). C1 and C6 indicate resonances of
galactose.
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Attempts to shift the GalCer C-6 resonance ( 61.361) using
paramagnetic shift reagents (Pr3+ and Yb3+)
yielded unsatisfactory results, leading us to adopt an alternate approach to quantify GalCer in the inner and outer vesicle leaflets of
SUVs. Our approach was based on using Mn2+ ions as a
bilayer-impermeant relaxing reagent to measure the distribution of
cholesterol in the inner and outer leaflets of lipid
vesicles.3 A similar strategy
has previously been used to compare the accessibility of cholesterol in
the outer leaflet of ester- and ether-linked phospholipid SUVs (39).
Titration experiments revealed that 5 mM Mn2+
was optimal for efficiently quenching the resonance at 61.361 ppm
derived from GalCer in the vesicle outer leaflet without affecting the
resonances of the inner leaflet. Because of the relatively low signal
intensity of the C-6 resonance of GalCer compared with the phospholipid
resonances in SUVs, natural abundance 13C NMR required that
the vesicles contain a relatively high content of GalCer (>20 mol %)
to achieve adequate signal-to-noise ratios to quantify the transbilayer
distribution of GalCer. To monitor GalCer transbilayer distribution
over a wide range of mole fractions, including those typical of
biological membranes, isotopic enrichment at the C-6 position of
galactose was deemed the best strategy to assure acceptable
sensitivity. Thus, we synthesized [6-13C]GalCer as
described in detail in the Supplemental Information and outlined in
Scheme 1. The resulting preparation was 99.8% isotopically enriched
and increased the intensity of the GalCer C-6 resonance almost 100-fold
(Fig. 1A).
Transmembrane Distribution of GalCer in POPC Vesicles--
To
determine GalCer distribution in phospholipid vesicles, POPC SUVs
containing 1 mol % [6-13C]GalCer were prepared and
analyzed by 13C NMR at 37 °C. Fig.
2 shows that the C-6 resonance of
galactose was well separated from POPC resonances and that its
signal-to-noise ratio (~20:1) was well suited for quantitative
analysis. Fig. 3 (left panel)
shows the 50-75-ppm region of the 13C NMR spectrum of the
POPC vesicles containing 1 mol % [6-13C]GalCer before
and after addition of 5 mM Mn2+. It is
noteworthy that GalCer strongly preferred the inner leaflet (Fig. 3,
right panel), with 70% of the GalCer molecules being inaccessible to Mn2+ ions (Table
I). The same high preference of
GalCer for the inner leaflet of POPC SUVs also was observed when the
GalCer content was increased to 2 mol% (Fig. 3, right
panel; Table I).
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Fig. 2.
13C NMR spectrum of
[6-13C]- GalCer-POPC vesicles. Inset B
shows an expanded region between 50 and 73 ppm.
CA, CB, and CC
indicate the resonances of the glycerol carbons, and C
and C indicate the phosphocholine headgroup resonances.
The POPC vesicles contained 1 mol % [6-13C]GalCer.
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Fig. 3.
Transbilayer distribution of GalCer in POPC
vesicles determined by 13C NMR. Left panel,
13C NMR spectra (55-75-ppm region) of POPC vesicles
containing 1 mol % [6-13C]GalCer, acquired in the
absence (lower spectrum), and in the presence of 5 mM Mn2+ (upper spectrum).
Right panel, transbilayer distribution of GalCer in POPC
vesicles containing either 1 or 2 mol % [6-13C]GalCer.
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Table I
GalCer, SPM, and POPC transbilayer distributions in SUVs
The inner and outer leaflet compositions of the vesicles were
calculated from NMR data acquired as described under "Experimental
Procedures." Units are expressed as mol %.
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To determine the transbilayer distribution of POPC in SUVs containing 1 or 2 mol % GalCer, 31P NMR measurements were performed in
the presence and absence of Pr3+ (see "Experimental
Procedures"). The outer-to-inner leaflet phosphorus ratios were 1.94 and 1.92, respectively, and were similar to the 1.90 ratio of pure POPC
SUVs (Table I).
SPM Alters the Transmembrane Distribution of GalCer--
To
investigate the effect of increasing SPM on the transbilayer
distribution of GalCer, SUVs with a constant amount of
[6-13C]GalCer and varying amounts of POPC and SPM
(e.g. 2:1, 1:1, or 1:2) were prepared. The transbilayer
distributions of [6-13C]GalCer and of each phospholipid
were then assessed by 13C and 31P NMR
spectroscopy, respectively. Fig. 4
(left panel) shows the 31P NMR spectra of
vesicles composed of equimolar POPC and egg SPM containing 1 mol % GalCer. As the top spectrum illustrates, the 31P resonances
of POPC ( 0.900 ppm) and SPM (0.246 ppm) were partially resolved
from each other in the absence of Pr3+, indicating
differing local environments for their phosphocholine headgroup
moieties. Addition of Pr3+ caused the POPC and SPM
resonances of the SUV outer leaflet to shift downfield, resulting in
the four distinct resonance peaks shown in the lower spectrum of Fig.
4. By comparison with SUVs containing different amounts of SPM and POPC
(e.g. 1:2 and 2:1), the SPM and POPC resonances were
assigned to the inner (0.246 and 0.900 ppm, respectively) and outer
(7.596 and 5.203 ppm, respectively) leaflets. The peak assignments
agree well with earlier reports (35-38). The larger
Pr3+-induced downfield shift of outer leaflet SPM (compared
with PC) and the distinct 31P resonances of SPM and POPC in
the absence of lanthanide ions were consistent with earlier findings
(32, 38, 40). Quantitative assessment indicated that the content of
phospholipid (both POPC and SPM) in the outer leaflet of SUVs far
exceeded that in the inner leaflet, consistent with the known mass
distribution of PC in SUVs of ~25-nm diameter (Fig. 4). However,
small but reproducible differences in the transbilayer distributions of
POPC and SPM could be distinguished (Table I). SPM showed a slightly
greater preference for the outer leaflet at the expense of POPC and
this tendency became more pronounced as the SPM mole fraction
increased. It is also noteworthy that the overall outside-to-inside
31P integrated signal ratios of the SPM/POPC vesicles
remained very close to the 2:1 ratio expected for SUVs at all SPM
compositions, consistent with the vesicles having average diameters
of ~25 nm (23, 25, 26). Previous studies have indicated that
sonication of SPM results in formation of unilamellar vesicles similar
in size to those generated by sonication of PCs (40).
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Fig. 4.
Transbilayer distribution of POPC and SPM
determined by 31P NMR. Left panel,
31P NMR spectra of vesicles comprised of equimolar POPC and
SPM and containing 1 mol % [6-13C]GalCer, acquired in
the absence (upper spectrum), and in the presence of 1 mM Pr3+ (lower spectrum). The
lower spectrum shows well resolved resonances derived from
POPC (a) and SPM (b) of the inner leaflet and
from POPC (c) and SPM (d) of the outer leaflet.
Right panel, transbilayer distribution of POPC
(A) and SPM (B) measured from 31P NMR
data for vesicles comprised of POPC/SPM at the molar ratios of 2:1,
1:1, and 1:2 and containing 1 mol % [6-13C]GalCer.
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Having established the transbilayer distribution of SPM and POPC when
mixed in SUVs, we next determined the effect of SPM on the transbilayer
distribution of 1 mol % [6-13C]GalCer by 13C
NMR (Fig. 5). We found that increasing
the SPM content in POPC SUVs shifted GalCer from the inner to the outer
leaflet. The amount of GalCer localized to the inner leaflet decreased
from 70% in pure POPC vesicles to only 40% in POPC/SPM (1:2)
vesicles. We believe that this represents the first evidence showing
that SPM can dramatically alter the transbilayer distribution of a
simple monohexosylceramide, such as GalCer, in phospholipid
vesicles.
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Fig. 5.
The effect of SPM on the transbilayer
distribution of GalCer determined by 13C NMR.
Left panel, 13C NMR spectra (50-80-ppm region)
of vesicles comprised of equimolar POPC and SPM and containing 1 mol % [6-13C]GalCer, acquired in the absence (lower
spectrum) and in the presence of 5 mM Mn2+
(upper spectrum). Right panel, transbilayer
distribution of GalCer in vesicles composed of POPC/SPM at the molar
ratios of 2:1, 1:1, and 1:2 and containing 1 mol % [6-13C]GalCer.
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DISCUSSION |
We have quantified the transbilayer distribution of GalCer in
phospholipid vesicles by 13C NMR spectroscopy. When used in
combination with 31P NMR approaches that monitor
phospholipid transbilayer distribution, the strategy provides novel
insights into the effect of changing SPM content on the transbilayer
distribution of simple glycosphingolipids. The results reveal two
notable findings. First, at low mole fractions, GalCer strongly prefers
the inner leaflet of POPC SUVs. Second, increasing the SPM
content of the POPC SUVs shifts the transbilayer distribution of GalCer
toward the outer leaflet. Ramifications of these observations are
discussed below.
In POPC SUVs containing low mole fractions of GalCer (1 or 2 mol %),
70% of the glycolipid is localized in the inner leaflet. This
corresponds to a doubling of the GalCer inner membrane concentration with respect to POPC, which is 34% localized in the inner leaflet (Table I). The mass distribution of PC in SUVs (1:2 inner
leaflet-to-outer leaflet) is well established (7-9). What is
remarkable about the transmembrane distribution of GalCer is its strong
preference for the inner leaflet when present at 1 or 2 mol % in POPC
SUVs. Earlier studies of PE and PG (see Introduction) revealed just the
opposite behavior in that these lipids strongly preferred the outer
leaflet of PC SUVs (29, 30). Only when present at 10 mol % (or more)
in PC SUVs did PE and PG assume transmembrane distributions that can be
rationalized by the structural parameters associated with their overall
shape, charge, and hydration (22, 29, 30). The preferential
localization of low mole fractions of PE and PG to the outer leaflet of
PC SUVs has been explained as a general lattice response linked to the
"looser" molecular packing of the outer leaflet of PC SUVs (30).
Our results clearly show that GalCer does not conform to the PE/PG
transmembrane localizations in fluid PC bilayers previously reported
for highly curved phosphoglyceride vesicles. This may be a consequence
of GalCer having a completely uncharged and moderately hydrated polar
headgroup compared with the zwitterionic or ionic headgroups of
phosphoglycerides (36, 41).
The second major finding of this study is that increasing the SPM
content of POPC SUVs dramatically shifts the transbilayer distribution
of GalCer toward the outer leaflets. This shift occurs even though SPM
and POPC have chemically identical phosphocholine headgroups. Not
surprisingly, our 31P NMR measurements in the presence and
absence of the paramagnetic shift ion, Pr3+, show that SPM
and POPC localize quite similarly in SUVs with SPM showing only a
slight preference for the outer leaflet (38, 40). However, the impact
of increasing SPM content on GalCer transmembrane distribution is clear
and dramatic (Table I). It is likely that the remarkable shift in the
transbilayer localization of GalCer toward the outer leaflets of
SPM/POPC SUVs reflects changes in the in-plane interactions that occur
between GalCer and SPM relative to those that occur between GalCer and
POPC in highly curved vesicles. The structural features of GalCer, SPM, and POPC likely to play a role in this behavior are the following. First, consider the lipid hydrocarbon region. POPC has the naturally prevalent PC motif consisting of sn-1 chain saturation and
sn-2 chain unsaturation. Both egg SPM and
[6-13C]GalCer have the naturally prevalent sphingolipid
motif consisting of sphingosine and a saturated acyl chain. Compared
with POPC's oleoyl chains, the mostly palmitoyl acyl chains (~85%)
of egg SPM would be expected to pack better with the palmitoyl chains of [6-13C]GalCer. With regard to the polar headgroup
region, one might mistakenly assume no difference among PC and SPM
because of the chemical identity of their phosphocholine headgroups.
However, it is clear that, when PC and SPM are mixed together in SUVs, the 31P NMR resonances of these lipids display different
chemical shifts, indicating that the local environments near their
respective phosphate groups are not identical. One explanation may be
that SPM, but not PC, forms intramolecular hydrogen bonds involving the
hydroxyl group at carbon 3 of the sphingoid base and either the bridge oxygen or ester oxygen of phosphate (40, 42-44). This capability may
contribute to the metastable behavior and different structural conformations known to occur in SPMs (Refs. 43-46 and references therein). In addition, the ceramide region of SPM and GalCer contain amide-linked acyl chains, which are thought to participate in intermolecular hydrogen bonding lattices via bridging water molecules, as well as 4,5-trans double bonds that further modulate intermolecular interactions (10, 42). The combined differences in the headgroup and
interfacial regions of SPMs and PCs appear likely to affect their
interactions with GalCer. Altogether, our results emphasize that subtle
structural and conformational changes to the interfacial zone of
bilayer matrix lipids, such as PC and SPM, can significantly affect the
transbilayer distribution of simple sphingolipids, such as cerebroside,
in curved membranes.
Physiological Relevance and Implications--
Lipid transmembrane
asymmetry is of fundamental importance to the health of cells, and a
loss of this asymmetry has severe detrimental effects. During late
apoptotic events as well as under many other pathological conditions
such as diabetes, malaria, and sickle cell disease, a loss of
phosphatidylserine (PS) asymmetry occurs (47). A defect in the
aminophospholipid translocase or activation of the phospholipid
scramblase causes abnormal exposure of PS on the exoplasmic leaflet
from its normal cytoplasmic orientation (48-50). PS externalization
during the lipid scrambling process has recently been linked to the
inward translocation of external SPM (6). This "flopped" SPM pool
is hydrolyzed by cytosolic sphingomyelinase to ceramide as part of the
execution phase of apoptosis. SPM depletion from the plasma membrane
leads to a redistribution of cholesterol to intracellular sites and/or
the efflux of cholesterol to external acceptors such as serum
lipoproteins and cyclodextrins (51, 52). An important consequence of
SPM transmembrane migration and associated ceramide generation is a
triggering of membrane destabilization and an increase in membrane
fission processes involving membrane blebbing and vesicle shedding.
Other striking examples implicating sphingolipid transmembrane
distributions in the triggering of membrane vesiculation in cell and
model membranes also have been reported (1, 53).
Gaining insights into the effects of high curvature on membranes is an
area of increasing interest in cell biology because of the importance
of membrane fission events in generating transport vesicles (54). The
ability of endophilin, a presynaptically enriched protein that binds
the GTPase dynamin and synptojanin, to generate very highly curved
membrane tubules underscores the potential importance of
tubulovesiculation processes to membrane trafficking events in the cell
(55). Investigations of sphingolipid transbilayer distributions in
curved membranes of defined composition, such as those reported here,
are likely to provide a valuable foundation for a better understanding
of cellular processes initiated by or utilizing curved membrane regions
where sphingolipids are important.
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FOOTNOTES |
*
This work was supported by the Academy of Finland (to
P. M.); NHLBI, National Institutes of Health (NIH) Grant 16660 (to
R. B.), NIH Grant RR-04654 (to W. J. B.), NIGMS, NIH
Grant 45928 (to R. E. B.); and by The Hormel
Foundation. This investigation was presented in part at the
Phospholipid Membrane Structure Platform of the
45th Biophysical Society Annual Meeting
held in Boston, MA, February 2001 (Mattjus, P., Valiyaveettil,
J. T., Malewicz, B., Bittman, R., Baumann, W. J., and Brown, R. E.
(2001) Biophys. J. 80, 331a).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.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Information and Scheme 1.
§
These authors contributed equally to this study.
To whom correspondence should be addressed: The Hormel
Inst., University of Minnesota, 801 16th Ave. NE,
Austin, MN 55912. Tel.: 507-433-8804; Fax: 507-437-9606; E-mail:
rebrown@hi.umn.edu or reb@tc.umn.edu.
Published, JBC Papers in Press, March 21, 2002, DOI 10.1074/jbc.M201305200
2
Refs. 56-60 pertain to Supporting Information
that describes the complete synthesis of [6-13C]GalCer
and that can be accessed on-line at the JBC web site.
3
B. Malewicz and W. J. Baumann, unpublished data.
| |
ABBREVIATIONS |
The abbreviations used are:
PC, phosphatidylcholine;
SUVs, small unilamellar vesicles;
PE, phosphatidylethanolamine;
PG, phosphatidylglycerol;
GalCer, galactosylceramide;
POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;
SPM, sphingomyelin;
PS, phosphatidylserine.
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ABSTRACT
INTRODUCTION