Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells

Article abstract of DOI:10.1194/jlr.M001974

Human fi broblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [³ H] choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([³ H]SM-N ₃). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fi-broblasts resulted as highly enriched in [³ H]SM-N ₃. However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fi broblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specifi c lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions. Copyright

Full text of DOI:10.1194/jlr.M001974

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Photoactivable sphingosine as a tool to study membrane
microenvironments in cultured cells
Massimo Aureli, Simona Prioni, Laura Mauri, Nicoletta Loberto, Riccardo Casellato,
Maria Grazia Ciampa, Vanna Chigorno, Alessandro Prinetti, and Sandro Sonnino1
Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on
Neurodegenerative Diseases, University of Milano, 20090 Segrate, Italy
Abstract Human fibroblasts from normal subjects and
Niemann-Pick A (NPA) disease patients were fed with two
labeled metabolic precursors of sphingomyelin (SM), [3H]
choline and photoactivable sphingosine, that entered into
the biosynthetic pathway allowing the synthesis of radioac-
tive phosphatidylcholine and SM, and of radioactive and
photoactivable SM ([3H]SM-N3). Detergent resistant mem-
brane (DRM) fractions prepared from normal and NPA fi-
broblasts resulted as highly enriched in [3H]SM-N3. However,
lipid and protein analysis showed strong differences between
the two cell types. After cross-linking, different patterns of
SM-protein complexes were found, mainly associated with
the detergent soluble fraction of the gradient containing
most cell proteins. After cell surface biotinylation, DRMs
were immunoprecipitated using streptavidin. In conditions
that maintain the integrity of domain, SM-protein complexes
were detectable only in normal fibroblasts, whereas disrupt-
ing the membrane organization, these complexes were not
recovered in the immunoprecipitate, suggesting that they in-
volve proteins belonging to the inner membrane layer.
These data suggest that differences in lipid and protein com-
positions of these cell lines determine specific lipid-protein
interactions and different clustering within plasma mem-
brane. In addition, our experiments show that photoactiva-
ble sphingolipids metabolically synthesized in cells can be
used to study sphingolipid protein environments and sphin-
golipid-protein interactions.—Aureli, M., S. Prioni, L. Mauri,
N. Loberto, R. Casellato, M. G. Ciampa, V. Chigorno, A.
Prinetti, and S. Sonnino. Photoactivable sphingosine as a
tool to study membrane microenvironments in cultured
cells. J. Lipid Res. 2010. 51: 798–808.
the membrane also enriched in cholesterol, dipalmi-
toylphosphatidylcholine, and proteins necessary for the
cell signaling processes (1). Much information is available
on the composition of membrane lipid domains of cells in
culture (2, 3) and tissues (4). On the other hand, there is
somewhat scant information on how lipids are capable of
modulating protein functions and on the distribution of
components within the two membrane layers. A possible
and widely accepted working hypothesis on this latter
point is that the fine tuning of receptor functions is
achieved by dynamic lateral interactions with SLs (5).
These interactions would induce conformational changes
in the receptors, directly (e.g., changing its intrinsic ty-
rosine kinase activity) (6, 7) or indirectly (e.g., changing
its association with regulator or substrate proteins) (8),
thus affecting their biological functions. As SLs, gly-
cosphingolipids (GSLs) attracted the interest of scientists
and many papers are available on membrane ganglioside-
protein interactions and on the regulatory effect exerted
by gangliosides on several membrane proteins (5). In ad-
dition, in recent years, sphingomyelin (SM) was suggested
to have a specific role in membrane organization (9, 10).
In addition, the availability of a membrane SM synthase
besides membrane sphingomyelinases suggests the exis-
tence of a physiological process capable of modifying the
SM:ceramide ratio at the membrane level and, as a conse-
quence, some membrane properties like membrane cur-
vature and membrane organization, according to the
necessity of the cell (11).
Supplementary key words gangliosides • lipid rafts • membranes •
Niemann-Pick disease • sphingolipids • sphingomyelin • photo-
labeling
Studies on ganglioside-protein environment and gangli-
oside-protein interactions were performed in the past by
Abbreviations: DRM, detergent-resistant membrane; EMEM, Mini-
mal Essential Medium with Earle’s salt; GSL, glycosphingolipid; HD,
high density; IP, immunoprecipitate; NPA, Niemann-Pick type A; PC,
phosphatidylcholine; PVDF, polyvinyldifluoride; SL, sphingolipid; SM-
N3, photoactivable SM; [3H]SM-N3, radioactive and photoactivable SM;
Sph, sphingosine; Sph-N3, photoactivable sphingosine; Sph-NH2, re-
duced photoactivable sphingosine; THF, tetrahydrofurane.
1 To whom correspondence should be addressed.
Sphingolipids (SLs) are cell membrane components
highly enriched in lipid domains, which are portions of
This work was supported by Mizutani Foundation for Glycoscience Grant
070002 to A.P. and Associazione Italiana Ricerca sul Cancro (AIRC 2006-
2008) and Cariplo Foundation Grants to S.S.
Manuscript received 2 October 2009.
e-mail: sandro.sonnino@unimi.it
The online version of this article (available at http://www.jlr.org)
contains supplementary data.
Published, JLR Papers in Press, October 10, 2009
DOI 10.1194/jlr.M001974
Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.
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were solubilized in this order in 230 ␮l of dry dichloromethane
loading the cell plasma membranes with photoactivable
and tritium labeled compounds that after illumination,
form covalent linkages with closely locating proteins
(12–14). As a result, radioactive ganglioside-protein com-
plexes could be isolated and analyzed.
in a three neck flask provided with an N2 septum-inlet adaptor
and a condenser. The reaction mixture was refluxed for 3 h
under continuous stirring, then cooled and evaporated under
vacuum. The product was purified by flash chromatography
with silica gel 60 column equilibrated and sequentially eluted
with hexane/EtOAc, 24:1 by vol and hexane/EtOAc 1:4 (90%
yield).
To a solution of 210 ␮moles of sphingosylphosphorylcholine
(Sigma) in 2.5 ml of chloroform:methanol, 1:2 by vol, photoacti-
vable 12-aminolauric acid pentafluorphenylester, 1-hydroxyben-
zotyriazole (Sigma), and tributylamine in the molar ratio of
1.1:1.8:1.5 with respect to fatty acid derivative, were added. After
vigorous stirring for 12 h at room temperature, the reaction mix-
ture was dried and SM-N3 purified by a silica gel 60 flash column
(1 × 15 cm), equilibrated, and eluted with chloroform/metha-
nol/water, 70:25:3 by vol.
To a solution of 100 ␮moles of SM-N3 in 4 ml of tetrahydro-
furane (THF) and 1ml of 100 mM Tris-HCl, pH 7.4, dithiothre-
itol (Sigma) was added to a 200 mM final concentration. The
reaction mixture was stirred for 48 h at room temperature and
the SM-NH2 synthesized was purified on C-18 reverse-phase silica
gel flash column (1 × 15 cm), equilibrated and eluted with meth-
anol/water/triethylamine, 15:2:1 by vol (22). Structural analysis
of SM-NH2 was performed by mass spectrometry.
Loading the plasma membrane with photoactivable SM
results in difficulties due to the amphiphilic properties of
the molecule. In fact, SM is much more hydrophobic than
gangliosides and in aqueous solution, forms large aggre-
gates that are in equilibrium with a very scant number of
monomers. Only monomers become components of the
membranes, whereas the large aggregates are phagocyted
and then catabolyzed in the lysosomes (15, 16). To over-
come this and to study SM-protein interactions, cultures of
human fibroblasts prepared from normal subjects and
from Niemann-Pick type A (NPA) disease patients were
fed with photoactivable sphingosine and tritiated choline,
both precursors for the biosynthesis of [3H]SM-N3. NPA
disease is a genetic SM lysosomal storage disease due to
the lack of acid sphingomyelinase activity (17). These cells
have a different SM content and make it possible to study
the role of the excess of SM in membrane organization.
Cell proliferation and viability after Sph and Sph-N3
treatment
MATERIALS AND METHODS
A total of 25 × 103 cells of normal or NPA fibroblasts were
plated in 60 mm cell culture plates. After 6 h, the cells were
treated with 38 nM of Sph or Sph-N3 for up to 72 h. After 24, 48,
and 72 h, cells were detached with trypsin, incubated in 0.25%
Trypan blue solution for 2 min, and counted using a Bürker
chamber. The dead cells were determined to be Trypan blue-
positive cells.
Cell cultures
Normal human skin fibroblasts were obtained by the punch
technique from normal children (age 6–12 months), cultured
and propagated as described (18) in 60 mm dishes (0.42 0.10
mg protein/dish), using Minimal Essential Medium with Earle’s
salt (EMEM) supplemented with 10% FBS. Pathological human
fibroblasts, provided by Istituto Nazionale Neurologico “Carlo
Besta”, were prepared as above from a 7-year-old NPA patient.
Pathological cells had an acidic sphingomyelinase activity on the
artificial substrate 2-N-hexadecanoylamino-4-nitrophenylphos-
phorylcholine lower than 4% and a 6-fold increase of SM with
respect to control cells. For the experiments, cells were used at
confluence.
Feeding of cells with Sph-N3 followed by [3-3H(Sph)]GM1
or [3-3H(Sph)]SM administration
Confluent 60 mm cell plates of normal and NPA fibroblasts
were shifted in a dark room under red safelight and incubated for
2 h with 38 nM Sph-N3 solubilized in EMEM containing 10% FBS.
[3-3H(Sph)]GM1 (1.2 Ci/mmol) or [3-3H(Sph)]SM (0.375 Ci/
mmol) (23) were solubilized in an appropriate volume of pre-
warmed (37°C) EMEM to obtain a Sph concentration of 5 × 10Ϫ7
M and 15 × 10Ϫ7 M, respectively. The medium from normal and
NPA fibroblasts, fed or not with Sph-N3, was removed and the
cells rapidly washed with EMEM. A total of 2 ml of the medium
containing the radioactive lipids was added to each plate and the
cells were incubated for 4 h pulse at 37°C. After pulse, cells were
washed twice with serum-free culture medium and then main-
tained in normal cell culture medium for 15 h chase. Cells were
washed twice with PBS and harvested in water. After lyophiliza-
tion, cells were submitted to lipid extraction. The incorporation
of radioactivity was determined by liquid scintillation counting
and the radioactive lipids were separated by high-performance
thin-layer chromatography (HPTLC) with the solvent system
CHCl3/CH3OH/(CH3)2CHOH/0.2% aqueous CaCl2, 20:60:20:4
by vol and analyzed by radioimaging.
Synthesis of Sph-N3
Sphingosine (Sph) derivative, containing the amine group at
the terminal carbon 12 (Sph-NH2), was prepared as reported
(19–21). The nitrophenylazide was then coupled to the N-termi-
nal group and the protection group was removed in order to ob-
tain photoactivable sphingosine (Sph-N3) (Fig. 1A). The reactions
details are described in the supplementary data.
Synthesis of photoactivable SM (SM-N3) and its reduced
form SM-NH2
SM containing a photoactivable fatty acid chain (see Fig. 1B)
was synthesized under red safelight. A total of 286 ␮moles of
12-aminododecanoic acid (Merck GmbH) were dissolved in 5 ml
of anhydrous dimethylformamide. An equimolar quantity of tri-
ethylamine and a 2-fold molar quantity of 4-F-3-NO2-phenylazide
(Sigma), dissolved in 5.8 ml of anhydrous methanol were added.
The reaction was stirred overnight at 80°C. The mixture was
dried and the photoactivable fatty acid was purified by two flash
chromatographic columns (silica gel 60, hexane/EtOAc, 1:2 by
vol and silica gel 60, hexane/EtOAc, 2:1 by vol) (80% yield).
2-Chloro-1-methylpyridinium iodide (Sigma), pentafluoro-
phenol (Merck GmbH), photoactivable 12-aminododecanoic
acid, and dry tributylamine in the molar ratio of 1.2:1.1:1:2.4
Feeding of cells with Sph-N3 and [methyl-3H]choline
Confluent cell cultures were shifted in a dark room under red
safelight and incubated for 2 h with 38 nM Sph-N3 solubilized in
EMEM containing 10% FBS. Cells were then maintained in se-
rum-free EMEM for 12 h and in serum-free EMEM containing 40
␮M zinc chloride for other 12 h (24). Then cells were fed with 56
␮M [methyl-3H]choline (Amersham) solubilized in serum-free
Membrane sphingomyelin protein environment
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Fig. 1. Chemical structures. A: Photoactivable sphingosine derivate, Sph-N3. B: Photoactivable sphingomyelin, SM-N3. C: Reduced form
of SM-N3, SM-NH2.
EMEM for 12 h. Finally medium was substituted with EMEM con-
taining 10% FBS for the last 12 h of incubation.
Preparation of DRM fractions by sucrose gradient
centrifugation
After photolabeling and biotinylation, cells were subjected to
homogenization and ultracentrifugation on discontinuous su-
crose gradient, as previously described (2). Briefly, cells were
harvested, lysed in 1% Triton X-100 in TNEV (10 mM Tris-HCl
buffer, pH 7.5, 150 mM NaCl, 5 mM EDTA) in the presence of
1 mM Na3VO4, 1 mM PMSF, and 75 mU/ml aprotinin, and
Dounce homogenized (10 strokes, tight). Cell lysate (1 mg of
cell protein/ml) was centrifugated 5 min at 1,300 g to remove
nuclei and cellular debris. The postnuclear fraction was mixed
with an equal volume of 85% sucrose (w/v) in TNEV, placed at
the bottom of a discontinuous sucrose gradient (30–5%), and
centrifugated for 17 h at 200,000 g at 4°C. After ultracentrifuga-
tion, 11 fractions were collected starting from the top of the
tube. Equal amounts of the low density fractions 4, 5, and 6
Cell surface biotinylation and cell illumination
Cells fed with Sph-N3 and [methyl-3H]choline were washed five
times with culture medium containing 10% FBS and twice with
PBS. Cells were then maintained for 1 h in serum starvation,
washed twice with PBS, and incubated with 0.25 mg/ml of sulfo-
NHS-biotin (Pierce) in PBS, pH 7.4 (5 ml/dish) for 30 min at
4°C (25). Under these experimental conditions, the internaliza-
tion of the biotin derivative does not occur and biotinylation is
restricted to the cell surface (26). After biotin labeling, cells were
rinsed twice with ice-cold PBS, 4 ml of ice-cold PBS were added,
and cells were illuminated for 45 min under ultraviolet light
(␭=360 nm) (27–30). Cell viability was assessed by the trypan blue
exclusion method (31).
Fig. 2. Experimental model scheme. Sph-N3 and [methyl-3H]choline were administered to human fibro-
blasts in culture with the aim of providing cells with photoactivable and radioactive precursors able to enter
the sphingolipid metabolic pathways, allowing the production of radioactive and photoactivable sphingomy-
elin able to interact with neighboring proteins and to form, after cell illumination, stable radioactive SM-
protein complexes.
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were put together to obtain the detergent-resistant membrane
samples were treated with 1% SDS in lysis buffer at 100°C for 5
min, then diluted 10-fold with lysis buffer (0.1% SDS final con-
centration) and reimmunoprecipitated as described previously,
obtaining a new IP. These conditions (domain-disrupting con-
ditions) are known to break up the membrane organization and
to allow the disaggregation of the DRM domains (34).
(DRM) fraction, whereas equal amounts of the high density
(HD) fractions 10 and 11 were put together to obtain the HD
fraction. The entire procedure was performed at 0–4°C in ice
immersion.
Immunoprecipitation experiments
Analysis of protein patterns
A total of 300 ␮l of DRM fraction containing 5–10 ␮g of pro-
teins was immunoprecipitated with 50 ␮l of streptavidin-coupled
magnetic beads (Dynal) previously washed twice with PBS. The
mixtures were stirred overnight at 4°C, then the immunopre-
cipitate (IP) was recovered by centrifugation (32). Under these
conditions (domain-preserving conditions), we preserved the
organization of lipid domains (32, 33). In some experiments, IP
Total cell homogenate, sucrose gradient fractions, IP, and su-
pernatant remaining after immunoprecipitation were analyzed
by SDS-PAGE. Total protein patterns were determined by silver-
staining directly to the gel, according to manufacturer’s instruc-
tions. Radioactive proteins due to the cross-linkage with [3H]
SM-N3 were analyzed by digital autoradiography after SDS-PAGE
Fig. 3. Effect of normal and photoactivable sphingosine on proliferation and viability of cultured fibroblasts. Six hours after seeding, NPA
and control fibroblasts were treated with normal (gray dotted line) or photoactivable (black dotted line) sphingosine solubilized in cell
culture medium, both at the final concentration of 38 nM for up to 72 h. The proliferation and viability were compared with untreated cells
(gray line). After 24, 48, and 72 h, the number of total cells (A1, B1) and of dead cells (A2 and B2, left) was evaluated by Trypan blue exclu-
sion assay as described in Materials and Methods. Data are the means SD of three different experiments.
Membrane sphingomyelin protein environment
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glycerophospholipids from the organic phases breaking their es-
ter bonds, and maintaining unaltered the amide linkage of sphin-
golipids. The reaction was blocked by adding 120 ␮l 0.5 M HCl in
CH3OH.
Finally, after phase separation (by adding 1 ml of CHCl3/
CH3OH/H2O, 70:18:12 by vol), the new organic phases were
loaded on HPTLC plates and run in CHCl3/CH3OH/CH3-
COOH/H2O, 30:20:2:1 by vol. SM was detected by spraying the
TLC with a molybdate reagent (36).
Other analytical methods
Radioactivity associated with cells, medium, gradient fractions,
and lipid extracts was determined by liquid scintillation count-
ing. The protein content was determined according to Lowry et
al. (37) with BSA as reference standard. Mass spectrometry was
carried out on a ThermoQuest Finnigan LCQDECA ESI-MS and a
Xcalibur™ data system. Samples were dissolved in CH3OH at a
concentration of 20–200 ng/␮l and introduced through HPLC
instrument. Ionization was performed under the following con-
ditions: spray voltage, 4Kv; sheath gas flow rate, 50 arbitrary units;
capillary temperature, 260°C; capillary voltage, -42 V. The scan-
ning range was m/z 200–1600, and fragmentation voltage for
collision-induced dissociation was 25–90%.
Fig. 4. Radioactive lipids analysis of normal (lanes 1, 2, 3, 4) or
NPA (lanes 5, 6, 7, 8) cells treated (lanes 1, 3, 5, 7) or not (lanes 2,
4, 6, 8) with Sph-N3 and fed with [3-3H(Sph)]GM1(lanes 1, 2, 5, 6)
or [3-3H(Sph)]SM (lanes 3, 4, 7, 8). One thousand dpm of the total
lipids extracted from normal and NPA fibroblasts fed with Sph-N3
followed by radioactive GM1 or SM administration were separated
by HPTLC using CHCl3/CH3OH/(CH3)2CHOH/0.2% aqueous
CaCl2, 20:60:20:4 by vol. as solvent system. Radioactive lipids were
detected by digital autoradiography performed with a Biospace
␤-imager instrument for 48 h.
Statistical analysis
Experiments were run in triplicate, unless otherwise stated.
Data are expressed as mean value SD.
RESULTS
Chemical synthesis
and blotting on a polyvinyldifluoride (PVDF) membrane. Biotiny-
lated proteins were recognized with horseradish peroxidase-con-
jugated streptavidin (Vector) and enhanced chemiluminescence
detection (Pierce Supersignal).
We synthesized Sph-N3, and SM-N3 reported in Fig. 1. In
order to obtain Sph-N3 (Fig. 1A), the nitrophenylazide
group was coupled to the N-terminal group of Sph-NH2 pre-
pared as reported (19–21) with some modifications in order
to increase the final yield. The reactions details are de-
scribed in supplementary data. The synthesis of SM-N3 has
been developed for the first time. The two products were
Lipid analysis
Aliquots of cell homogenate, low density (4, 5, 6, ) and high
density fractions (10, 11,) were dialyzed and lyophilized. These
samples were subjected to lipid extraction with CHCl3/CH3OH/
H2O, 2:1:0.1 by vol (35). The total lipid extract was analyzed by
thin layer chromatography (HPTLC Kieselgel 60, 10 × 10 cm)
with the solvent system CHCl3/CH3OH/0.2% aqueous CaCl2,
50:40:8 by vol. The HPTLC visualization was obtained by digital
autoradiography and the identification of lipids was accom-
plished by comigration with standard lipids, the lipid mixture
characterization having been previously established (2).
To verify the biosynthesis of [3H]SM-N3, cells fed with [methyl-3H]-
choline and Sph-N3 were subjected to detergent homogeniza-
tion, sucrose gradient fractionation, and lipid analysis under red
safelight. Then, again under red safelight, the total lipid extracts
obtained from cell homogenate, DRM, and HD fractions were
dried and dissolved in 40 ␮l of THF. 10 ␮l of 100 mM Tris-HCl
containing 200 mM dithiothreitol were added and the reaction
mixture was stirred for 72 h at room temperature. Radioactive
SM-NH2 was analyzed by HPTLC performed with two sequential
runs in CHCl3/CH3OH/H2O, 50:40:8 by vol, followed by digital
autoradiography.
Aliquots of the total lipid extracts from cell homogenates, DRM,
and HD fractions were subjected to a two-phase partitioning (35).
The organic phases were dried under nitrogen flow and resus-
pended with 100 ␮l of CHCl3 and 100 ␮l of 0.6 M NaOH in
CH3OH and allowed to stand at 37°C for 3 h and overnight at
room temperature. This alkaline treatment allows for removal of
Fig. 5. Radioactive lipids analysis. TLC separation of the total lip-
ids extracted from normal (lane A) and NPA (lane B) fibroblasts
fed with Sph-N3 and [methyl-3H]choline. One thousand dpm were
applied on a 4 mm line for each sample. TLC was run in CHCl3/
CH3OH/0.2% aqueous CaCl2, 50:40:8 by vol. Digital autoradiogra-
phy was performed with a Biospace ␤-imager instrument for 48 h.
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98% homogeneous and remained stable when stored in
methanol at –20°C in a container protected from the light.
catabolism also produced Sph, a part of which was recycled
for the biosynthesis of SM (lane 1–2). In NPA fibroblasts,
the metabolism of GM1 was lower than in normal cells,
due to the lysosome impairment, but resulted in the same
lipid pattern (lane 5–6). As expected, the radioactive SM is
largely converted into Cer in normal fibroblasts (lane 3–4)
(with some recycling of Sph for the biosynthesis of com-
plex SLs), whereas this does not occur or is a very minor
process in NPA fibroblasts (lane 7–8) due to the lack of
the activity of lysosomal sphingomyelinase.
Effect of the Sph-N3 administration on normal and NPA
fibroblasts
Normal and NPA fibroblasts were fed with Sph-N3 (Fig.
2) in order to verify cell toxicity. As shown in Figure 3,
neither normal nor NPA fibroblasts present alterations in
cell proliferation and death in the presence of both Sph
and Sph-N3.
Feeding with Sph-N3 does not alter the capability of cells
to take up GM1 or SM and insert them into the SL meta-
bolic pathway. We had overlapping qualitative and quanti-
tative results of radioactivity incorporation, lysosomal
catabolism, and trafficking related to the recycling of
sphingosine in normal and NPA fibroblasts treated or not
with Sph-N3. The SL pattern obtained from cells treated
with Sph-N3 was comparable and almost identical to that
obtained from the corresponding control cells (Fig. 4). As
shown in Fig. 4, the radioactive GM1 incorporated in nor-
mal fibroblasts is catabolyzed to GM2, GM3, and Cer. The
[3H]SM-N3 biosynthesis
We verified that the endogenous SM content in NPA fi-
broblasts was strongly increased with respect normal cells:
109 versus 16 nmoles/mg cell proteins. Sph-N3 and
[methyl-3H]choline were administered to NPA and normal
fibroblasts in culture with the aim to give cell precursors
for the biosynthesis of tritiated and [3H]SM-N3, as ex-
plained in Fig. 2. To follow the [3H]SM-N3 distribution
within gradient fractions, cell homogenates were prepared
from fibroblasts fed under red safelight with the labeled
Fig. 6. Gradient fractions analysis. Gradient frac-
tions distribution (abscissa axis) of total radioactivity
(A1, B1), radioactive sphingomyelin (A2, B2), and
cell proteins (A3, B3) in normal and NPA cells. Data
are the means SD of three different experiments.
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precursors and were then loaded on a sucrose gradient.
The 11 fractions obtained by ultracentrifugation were
maintained in a dark room and subjected to lipid analysis.
Both cell lines incorporated an equivalent amount of ra-
dioactivity that was mainly associated with phosphatidyl-
choline (PC) and SM, which are the two membrane lipids
having choline as head group. As shown in Figure 5, the
90% of the total radioactivity was associated with PC and
the remaining with SM. As shown in Figure 6, cell radioac-
tivity was largely associated with fractions 9–11 (panel A1
and B1), corresponding to the HD fractions that contain
membranes solubilized by the detergent and the higher
quantity of cell proteins (panel A3 and B3). Moreover,
some radioactivity was also associated with fractions 4–6
corresponding to the low density fractions that are resistant
to the detergent solubilization. TLC analysis of the lipids
extracts showed different contents of PC and SM in the 11
fractions (Fig. 7). PC was distributed within all fractions,
being much more abundant in the high density fractions
9–11, whereas SM was highly concentrated in the low den-
sity detergent-resistant fractions 4–6. In particular, as
shown in Fig. 6 (Panel A2 and B2), around 70% of SM was
associated with the fractions 4–6 in both cell lines. The
[3H]SM recognized on the TLC plates as a double spot
due to heterogeneity in the fatty acids content, is the sum
of [3H]SM and [3H]SM-N3, the two compounds displaying
a very similar chromatographic behavior. To analyze the
ratio between [3H]SM and [3H]SM-N3, fractions were sub-
jected to chemical reduction as described above, in order
to convert SM-N3 into SM-NH2, which has a different chro-
matographic behavior and can be separated from SM-N3.
Figure 8 shows the TLC separation of the radioactive lipids
from low density-detergent resistant fractions treated by
chemical reduction. After quantification of the digital au-
Fig. 8. [3H]SM-N3 analysis. TLC separation of radioactive total
lipid extracts obtained from DRM fractions prepared from normal
(A) and NPA (B) fibroblasts. A total of 1000 dpm of control lipid
extracts (lane 2) and 1000 dpm of lipid extracts previously sub-
jected to chemical reduction with dithiothreitol (lane 1) were ap-
plied on a 4 mm line. TLC was performed with two sequential runs
in CHCl3/CH3OH/H2O, 50:40:8 by vol. The radioactivity was de-
tected by digital autoradiography using a Biospace ␤-imager instru-
ment for 48 h.
toradiography, we calculated that the ratio between [3H]
SM and [3H]SM-N3 in DRM of both normal and NPA cells
was about 5:1.
This result confirms that cells fed with photoactivable
precursors are able to biosynthesize photoactivable SLs
and in particular, that the use of tritiated choline in com-
bination with Sph-N3 yields [3H]SM-N3.
Photolabeling and immunoprecipitation experiments
Normal and NPA fibroblasts fed with Sph-N3 and
[methyl-3H]choline were biotinylated, illuminated under ul-
traviolet light, and then 11 fractions were separated by ultra-
centrifugation of the cell homogenates loaded on a sucrose
gradient as described in Materials and Methods. Proteins
from total homogenates, low- and high-density sucrose gra-
dient fractions were separated by SDS-PAGE, blotted on
PVDF membranes, and submitted to radioimaging for rec-
ognition of the tritiated SM-protein complexes. Figure 9 (A,
B, lane 1) shows that many complexes are formed in the
cells after illumination, suggesting that many proteins are in
proximity of SM. Analysis of the sucrose gradient fractions
shows that these complexes belong to the HD detergent-
soluble fractions (Fig. 9, A, B, lane 2) that comprise those
membranes containing a minor portion of SM-N3 (about
30%) and 98% of cell proteins. Instead, radioactive proteins
were hardly detectable in the low density fractions (DRM)
enriched in SM and containing only 1.5–2% of the total cell
proteins. Table 1 reports data on the radioactivity associ-
ated with cell lipids and proteins. About 0.01% of total cell
radioactivity was associated with the proteins belonging to
the low-density detergent-resistant fractions prepared
from normal cells fed with radioactive choline and Sph-N3.
Fig. 7. Radioactive lipids distributions within gradient fractions.
TLC separation of radioactive total lipid extracts obtained from
sucrose gradient fractions 4, 5, 6, 10, and 11 prepared from normal
(A) and NPA (B) fibroblasts. One thousand dpm were applied on
a 4 mm line for each sample. TLC was run in CHCl3/CH3OH/0.2%
aqueous CaCl2, 50:40:8 by vol. Digital autoradiography was per-
formed with a Biospace ␤-imager instrument for 48 h.
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biotinylated protein patterns. Radioactivity was associated
with a few proteins in normal cells, the main with an ap-
parent molecular mass of about 70 kDa (panel A, lane 6).
Instead, no radioactive proteins could be detected in the
immunoprecipitate from NPA cells (panel B, lane 6). To
determine the membrane topology of the cross-linked
proteins, the IP from normal cells was solubilized in boil-
ing SDS solution and reimmunoprecipitated with strepta-
vidin. Under these conditions, the membranes were
solubilized by the detergent and then only biotinylated
proteins were immunoprecipitated, whereas the proteins
associated with the internal side of the plasma membrane
were recovered in the surnatant (Fig. 10A). Figure 10B
shows that under domain disrupting conditions, radioac-
tive proteins remained in the supernatant and were not
immunoprecipitated, suggesting that the SM-protein com-
plexes are formed with proteins belonging to the inner
layer of plasma membrane.
Fig. 9. Proteins cross-linked with [3H]SM-N3 from cell homoge-
nates (lane 1), HD fraction (lane 2) and DRM fraction (lane 3)
prepared from normal (A) and NPA (B) fibroblasts were separated
by 10% SDS-PAGE, blotted on a polyvinyldifluoride (PVDF) mem-
brane, and visualized by digital autoradiography for 120 h. Proteins
recovered in the immunoprecipitation experiments performed in
domain preserving conditions starting from aliquots of DRM frac-
tions prepared from normal (A) and NPA (B) fibroblasts were sep-
arated by 10% SDS-PAGE. The proteins were directly detected by
silver staining (lane 4) or blotted on a PVDF membrane and then
visualized by Western blot using HRP-streptavidin (lane 5) or by
digital autoradiography for 120 h (lane 6). Data are the means of
three different experiments.
DISCUSSION
Several lipid derivatives containing fluorescent, para-
magnetic, or photoactivable probes have been used in the
past to study lipid metabolism, traffic, subcellular localiza-
tion, and interactions with other molecules. The use of
these derivatives implies the need to demonstrate that
their behavior mimics as close as possible that of natural
endogenous compounds. In the past, we synthesized
radioactive and photoactivable gangliosides to study
ganglioside-protein interactions. These gangliosides were
administered to cells and taken up to become membrane
components (12, 13). In previous papers (8, 12, 27, 38),
we used photoactivable gangliosides and produced evi-
dence that these derivatives, once taken up by cells, un-
dergo metabolic processing and intracellular distribution
similarly to the natural compounds. Indeed, the most criti-
cal step in the use of these derivatives is the initial associa-
tion with cell membrane (38). Based on this experience,
we designed a novel sphingosine derivative (Fig. 1A) that
easily enters the cells and is subsequently used for the bio-
synthesis of complex SLs, including SM, carrying the pho-
toactivable group.
In a lipid membrane impoverished of proteins, [3H]SM-
N3 preferentially binds lipids, not proteins. In order to
have enough radioactivity associated with proteins, the
DRM fractions (prepared from cells previously submitted
to surface biotinylation) were subjected to immunopre-
cipitation with streptavidin-conjugated beads under condi-
tions that preserve the membrane domain integrity. For
both cell lines, we recovered more than 90% of biotiny-
lated proteins in the IP samples (data not shown). The IPs
were submitted to SDS-PAGE and analyzed for their con-
tent in total protein, biotinylated protein, and, by digital
autoradiography, for [3H]SM-N3-protein complexes (Fig.
9). Figure 9, lanes 4 and 5, shows that many proteins de-
tectable by silver or by streptavidin staining were present
in the IP in both normal and NPA cells (panels A, B, re-
spectively). Nevertheless, it is possible to observe qualita-
tive and quantitative differences between total and
Using this tool, we studied the SM-protein interactions
in normal cells and in pathological cells, NPA disease cells,
where SM accumulation could determine or influence the
SM protein environment.
We performed experiments to verify cell toxicity for the
Sph-N3. The cell proliferation and viability didn’t change
TABLE 1. Radioactivity associated with normal and NPA fibroblasts.
Lipid Radioactivity
Protein Radioactivity
Cell Radioactivity
DRM
HD
(dpm × 10Ϫ3
DRM
HD
)
NPA cells
Normal cells
15,158 242
18,040 330
2,268 35
2,868 41.8
12,857 220
15,061 264
0.0022 0.0005
1.984 0.011
148.940 5.06
179.080 7.48
DRM, detergent resistant membrane corresponding to the membrane low density fraction; HD, high density
detergent-soluble membrane fraction. Data are the means SD of three different experiments.
Membrane sphingomyelin protein environment
805

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able reduction procedure capable of converting the azide
group into an aminic group in quantitative yield. The SM-
NH2 produced (Fig. 1C), was characterized by mass spec-
trometry and used as chromatographic standard. By
reducing the azide to amine, SM and SM-NH2 could be
separated by TLC. Applying the reduction reaction to our
samples, [3H]SM and [3H]SM-NH2 could be separated
and resulted in a ratio of about 5:1. In addition to this, we
verified that the topology of [3H]SM-N3 corresponds to
that of [3H]SM; in fact, it was highly enriched into the low-
density detergent-resistant fractions. This suggests that
cells dilute Sph-N3 into the endogenous Sph and use it as
the natural product. Therefore, the biosynthesized [3H]
SM-N3 should mimic endogenous SM and can be consid-
ered a useful tool to recognize the protein SM environ-
ment through recognition of SM-protein complexes
formed after cell illumination. After illumination, the ra-
dioactivity present in the DRM fraction was largely associ-
ated with lipids whereas the radioactivity associated with
proteins was hardly detectable by digital autoradiography
after SDS-PAGE separations and blotting on a PVDF mem-
brane. The problem was resolved by concentrating the
plasma membrane lipid domains by cell surface protein
biotinylation followed by immunoprecipitation of the
DRM fractions using streptavidin-coupled magnetic beads
performed under domain preserving conditions. Surpris-
ingly, we found that SM-protein complexes were formed
in normal cells while no radioactive bands could be identi-
fied in the IP from NPA cells (Fig. 9, lanes 6). In addition
to this, several differences were recognized in the protein
patterns, determined by silver staining, and in biotinylated
proteins composition of the IP samples obtained from the
DRM fractions of the two cell lines (Fig. 9, lanes 4 and 5).
Thus, the lysosomal storage of SM in NPA cells influences
the protein environment of membrane SM. As reported
elsewhere, [3H]SM-N3 has the photoactivable group at the
end of the Sph moiety; in this way, when the molecule is
inserted into the membrane, it remains located at the cen-
ter of membrane lipid core. Therefore, after illumination,
glycosyl-phosphatidyl inositol-anchored proteins and
transmembrane proteins can form complexes with photo-
activated SM and also be biotinylated. Complexes formed
with protein belonging to the membrane inner layer can
also be formed, but these are distinguishable from the pre-
vious being not biotinylated.
Fig. 10. Topology of SM-protein complexes belong to detergent
resistant membrane (DRM) of normal cells. The IP sample ob-
tained in domain preserving conditions from DRM fraction of L40
fibroblasts was reimmunoprecipitated in domain disrupting condi-
tions. Proteins recovered in the IP100 (lane 2) and in supernatant
remaining after immunoprecipitation 100 (lane 1) were separated
by 10% SDS-PAGE and blotted on a PVDF membrane, which was
stained with HRP-streptavidin (A) and then visualized by digital
autoradiography to detect the radioactive SM-protein complexes
(B). Data are the means of three different experiments.
upon 72 h of Sph-N3 administration in either normal or
NPA fibroblasts nor following the membrane insertion
and endocytosis of radioactive GM1 and radioactive SM.
Therefore, we verified that the plasma membrane func-
tionality was not affected by Sph-N3 feeding.
Normal and NPA fibroblasts were fed with Sph-N3 and
radioactive choline that entered into the biosynthetic
pathway of SM allowing the metabolic synthesis of photo-
activable and radioactive SM ([3H]SM-N3), useful, after
cell illumination, to form SM-protein complexes (see the
scheme in Fig. 2). The nitrene group, formed by illumina-
tion of the azide linked to the end of ceramide moiety of
SM, preferentially binds lipids due to lipid-lipid interac-
tions occurring in the lipid core of membrane, whereas
binds proteins in minor quantity (39). This occurs particu-
larly in lipid domains where lipids are strongly enriched
with respect to proteins. Nevertheless, we were mainly in-
terested to see the SM protein environment in these do-
mains, membrane portions highly enriched in SLs and
cholesterol and often called DRM or “lipid rafts,” where SM
represents 60–70% of the total cell SM content. [methyl-3H]
choline was taken up by the cells and as expected entered
into the metabolic pathway of the choline-containing com-
pounds. In fact, both PC and SM were found to be radioac-
tive compounds, the first in much larger quantity (Fig. 5).
Sph administered to fibroblasts in culture is rapidly taken
up where its recycling is near 80% (39). Now, we show that
fibroblasts in culture are capable of taking up Sph-N3 and
inserting it into the SL biosynthetic pathway. In both nor-
mal and NPA cells, the administration of Sph-N3 in combi-
nation with [methyl-3H]choline allows the metabolic
synthesis of a photoactivable and tritiated SM. [3H]SM-N3
and [3H]SM displayed a very similar TLC behavior and
were recognized on the TLC plate as a double spot due to
heterogeneity of the fatty acid moiety. To distinguish the
[3H]SM-N3 from the [3H]SM, we synthesized a standard of
SM-N3 (Fig. 1B) and, on this standard, we developed a reli-
To understand the topology of proteins involved in the
SM-protein complexes, the IP from the low density frac-
tion prepared from normal cells previously submitted to
biotinylation was solubilized at 100°C with SDS. Under
these conditions, the membrane is disrupted and only
biotinylated proteins can be immunoprecipitated with
streptavidin. In this way, we found that the SM-protein
complexes were not immunoprecipitated and remained
in the supernatant. Thus, the complexes involve proteins
of the inner layer (Fig. 10B).
Altogether, our results suggest the following consider-
ations. Precursors of SLs probed with a radionuclide and a
photoactivable group enter in the SL metabolic pathways,
allowing the biosynthesis of radioactive and photoactiva-
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Journal of Lipid Research Volume 51, 2010

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factor receptor kinase activity and cell growth. J. Biol. Chem. 266:
10174–10181.
ble SLs that display a correct cell topology. In this work,
using tritiated choline and Sph-N3 in normal and patho-
logical cells, we were able to produce tritiated and photo-
activable SM. The [3H]SM-N3 was highly enriched, as and
together with the natural SM, into a low-density detergent-
resistant membrane fraction often named DRM or “lipid
rafts”. After illumination, SL-protein complexes are
formed. The position of photoactivated group at the end
of ceramide moiety of SM and the lipid-lipid interactions
occurring in the membrane lipid core greatly favor the re-
actions within the hydrophobic chains. In confirmation of
this, 99% of the total radioactivity associated with the cells
was linked to lipids. The formation of SM-protein com-
plexes requires proximity and direct interaction between
SM and the protein. Many complexes were found in both
normal and NPA fibroblasts. Nevertheless, the two pat-
terns were different, indicating that the storage of SM due
to the lack of acidic sphingomyelinase in NPA disease has
repercussions on the membrane organization of both lipid
and proteins. We did not find SM-protein complexes in
DRM from NPA cells and, in DRM from normal cells, only
a few were formed with acylated or very hydrophobic pro-
teins of the inner layer (13). This suggests that SM in lipid
rafts of NPA cells is largely or completely excluded from
the lipid environment of proteins, thus the photoactivable
probe is not enough closed to protein in order to yield a
covalent linkage.
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