Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells

Article abstract of DOI:10.1194/jlr.M055319

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed

Full text of DOI:10.1194/jlr.M055319

Direct interaction, instrumental for signaling processes,
between LacCer and Lyn in the lipid rafts of
neutrophil-like cells
Elena Chiricozzi,* Maria Grazia Ciampa,* Giuseppina Brasile,* Federica Compostella,*
Alessandro Prinetti,* Hitoshi Nakayama,† Roudy C. Ekyalongo,† Kazuhisa Iwabuchi,1,† Sandro
Sonnino,* and Laura Mauri*
Department of Medical Biotechnology and Translational Medicine,* University of Milan, Milano, Italy; and
Institute for Environmental Gender-Specific Medicine,† Juntendo University Graduate School of Medicine,
Chiba, Japan
Supplementary key words lactosylceramide • photoactivable sphingo-
lipids • C24 fatty acid chain • interdigitation
Abstract Lactosylceramide [LacCer; ␤-Gal-(1-4)-␤-Glc-(1-
1)-Cer] has been shown to contain very long fatty acids that
specifically modulate neutrophil properties. The interac-
tions between LacCer and proteins and their role in cell sig-
naling processes were assessed by synthesizing two molecular
species of azide-photoactivable tritium-labeled LacCer hav-
ing acyl chains of different lengths. The lengths of the two
acyl chains corresponded to those of a short/medium and
very long fatty acid, comparable to the lengths of stearic
and lignoceric acids, respectively. These derivatives, desig-
nated C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), were
incorporated into the lipid rafts of plasma membranes of
neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[3H]
LacCer-(N3), but not C18-[3H]LacCer-(N3), induced the
phosphorylation of Lyn and promoted phagocytosis. Incor-
poration of C24-[3H]LacCer-(N3) into plasma membranes,
followed by illumination, resulted in the formation of sev-
eral tritium-labeled LacCer-protein complexes, including
the LacCer-Lyn complex, into plasma membrane lipid rafts.
Administration of C18-[3H]LacCer-(N3) to cells, however,
did not result in the formation of the LacCer-Lyn com-
plex. These results suggest that LacCer derivatives mimic
the biological properties of natural LacCer species and
can be utilized as tools to study LacCer-protein interac-
tions, and confirm a specific direct interaction between
LacCer species containing very long fatty acids, and Lyn
protein, associated with the cytoplasmic layer via myristic/
palmitic chains.—Chiricozzi, E., M. G. Ciampa, G. Brasile,
F. Compostella, A. Prinetti, H. Nakayama, R. C. Ekyalongo,
K. Iwabuchi, S. Sonnino, and L. Mauri. Direct interaction,
instrumental for signaling processes, between LacCer and
Lyn in the lipid rafts of neutrophil-like cells. J. Lipid Res.
2015. 56: 129–141.
Glycosphingolipids (GSLs) are amphiphilic compo-
nents of the outer layer of plasma membranes that par-
ticipate in the transduction of information across the
membrane by determining the lateral organization of cel-
lular membranes and/or by modulating the function of
several classes of membrane-associated proteins (1). More
than 400 different oligosaccharide chains associated with
GSLs have been identified, but the number of existing
GSL molecular species is at least 10-fold higher, due to the
heterogeneity in their ceramide moiety (2, 3). These phys-
icochemical properties suggest that the molecular varieties
and expression patterns of GSLs reflect their biological
functions in each organism.
Although GSL-enriched lipid rafts have been implicated
in a number of important membrane events (4–6), the
molecular mechanisms by which GSLs mediate cell func-
tions remain unclear. One major issue is the association of
GSLs with signal transducer molecules localized to the cy-
tosolic side of the plasma membrane. Over the past several
years, we have attempted to clarify this issue using cells
of the human neutrophilic lineage (7). Lactosylceramide
[LacCer; ␤-Gal-(1-4)-␤-Glc-(1-1)-Cer] species containing
Abbreviations: Bu3N, tributylamine; CerS, ceramide synthase; DDQ,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DFP, diisopropyl fluoro-
phosphate; D-HL-60 cell, DMSO-treated human promyelocytic leukemia
cell; DMF, dimethylformamide; DRM, detergent-resistant membrane;
Et3N, triethylamine; fMLP, formyl peptide (N-formylmethionine-leucine-
phenylalanine); GD1b, Galb1-3GalNAcb1-4(NeuAca2-8NeuAca2-3)Galb1-
4Glcb1-1Cer; GM1, Galb1-3GalNAcb1-4(NeuAca2-3)Galb1-4Glcb1-1’-Cer;
GM3, NeuAca2-3Galb1-4Glcb1-1’-Cer; GSL, glycosphingolipid; H2O,
water; LacCer, lactosylceramide [␤-Gal-(1-4)-␤-Glc-(1-1)-Cer]; MeOH,
methanol; MsCl, methane sulfonyl chloride; PNS, post nuclear super-
natant; PVDF, polyvinylidene difluoride.
This study was supported in part by a grant-in-aid (S1201013, S1311011)
from the Foundation of Strategic Research Projects in Private Universities from
the Ministry of Education, Culture, Sports, Science, and Technology, Japan,
and by Matsumae Foundation (for K.I.). S.S. was supported by funds obtained
through performance in tariff of the Department of Medical Biotechnology and
Translational Medicine, University of Milan.
Manuscript received 6 October 2014 and in revised form 14 November 2014.
1 To whom correspondence should be addressed.
e-mail: iwabuchi@juntendo.ac.jp (K.I.)
Published, JLR Papers in Press, November 23, 2014
DOI 10.1194/jlr.M055319
Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 56, 2015
129

C24:0 and C24:1 fatty acids, or C24-LacCer, are compo-
nents of plasma membrane lipid rafts in human neutro-
phils and act as pattern recognition receptors responsible
for chemotaxis, phagocytosis, and superoxide generation
(5, 7, 8). These functions are highly dependent on the Src
family kinase, Lyn. The interaction of microorganisms
with LacCer activates Lyn, resulting in its phosphorylation,
a reaction considered the first step in the Lyn-mediated
immunological functions of human neutrophils (9). These
events can be easily reproduced experimentally by treating
neutrophils with anti-LacCer antibodies.
The LacCer contents in lipid rafts of human promyelocytic
leukemia HL-60 cells induced to differentiate into neutro-
philic cells by DMSO are similar to those of human neutro-
phils (7). However, the LacCer on plasma membranes of
these DMSO-treated human promyelocytic leukemia cells
(D-HL-60 cells) is more enriched in shorter fatty acids than
the LacCer of neutrophils (7). Treatment of D-HL-60 cells
with formyl peptide (fMLP; N-formylmethionine-leucine-
phenylalanine), but not anti-LacCer monoclonal antibody,
was found to induce Lyn phosphorylation and chemotactic
and superoxide-generating activities. Nevertheless, treatment
of D-HL-60 cells, which consisted mainly of C16:0-LacCer
and had quite low contents of C24-fatty acid chains, with
anti-LacCer antibodies induced chemotactic and superoxide
generating activities following cell loading with exogenous
C24-LacCer (7). Lyn knockdown by siRNA completely abol-
ished the effect of C24:1-LacCer loading on the LacCer-
mediated functions of D-HL-60 cells. These observations
suggested that C24-LacCer is specifically required for neutro-
phil properties and that long hydrophobic chains on LacCer
may be responsible for their direct interaction with Lyn, via
interdigitation with the acyl chains that allow the association
of Lyn with the cytosolic membrane layer.
Under illumination, nitrophenylazide generates the highly
reactive nitrene moiety, which can covalently bind to
nearby molecules.
MATERIALS AND METHODS
Mouse anti-LacCer monoclonal IgMs T5A7 (5) and Huly-m13
were purchased from Ancell (Bayport, MN). Rabbit anti-phospho-
Lyn IgG (Y396) was from Abcam (Cambridge, UK), anti-Lyn IgG
was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-
G␣i IgG was from Cell Signaling (Beverley, MA). Phycoerythrin-
conjugated anti-human CD11b and mouse IgGIK were from
eBioscience (San Diego, CA). All other monoclonal antibodies
were from BD Biosciences (San Jose, CA). DMSO, BSA, and
fMLP were purchased from Sigma-Aldrich Japan (Tokyo, Japan).
LacCer species with homogeneous fatty acid moieties were syn-
thesized as described (7).
HL-60 cells were maintained in culture in RPMI-1640 medium
supplemented with 10% FBS. To induce differentiation into
D-HL-60 cells, HL-60 cells were cultured for 8 days in the pres-
ence of 1.3% DMSO, with differentiation confirmed by CD11b
expression using flow cytometry analysis (21, 22).
Synthesis of tritium-labeled photoactivable LacCer, [3H]
LacCer(N3)
Tritium labeled and photoactivable LacCer containing an azide
group at the end of the acyl chain with total lengths corresponding
to stearic acid, C18-[3H]LacCer(N3), and lignoceric acid, C24-[3H]
LacCer-(N3), were synthesized according to the scheme shown in
Fig. 1. C18-[3H]LacCer-(N3) was synthesized from commercially
available 12-aminododecanoic acid (11, 12), whereas C24-[3H]
LacCer-(N3) was synthesized from 18-amino-octadecanoic acid,
compound 16, which had been synthesized according to the
scheme shown in Fig. 2.
Synthesis of 18-amino-octadecanoic acid (see Fig. 2). To 8 ml
of a suspension of 0.75 M octadecanedioic acid (compound 7) in
CH3OH was added 104 ␮l of 98% H2SO4, and the reaction mix-
ture was stirred overnight under reflux. The mixture was concen-
trated under vacuum, diluted with ethyl ether, and washed three
times with saturated Na2CO3 solution. The organic phase was
dried over Na2SO4 end evaporated under reduced pressure, re-
sulting in a 97% yield of compound 8 (23).
Photolabeling experiments have been performed using
radiolabeled and photoactivable lipids (10). A GM1 gan-
glioside containing tritium-labeled sialic acid and with an
azide at the end of the ceramide moiety was the first radio-
labeled ganglioside (11), followed by the synthesis of
other photoactivable gangliosides (12). Upon addition
to cultured cells, these exogenous ganglioside derivatives
become associated with the cells and undergo metabolic
processing in ways that closely resemble those observed for
natural compounds, suggesting that they may be utilized
as tools to study the interactions between GSLs and pro-
teins. Photolabeling with photoactivable GM3 provided
evidence for the specific role of GM3 in reducing insulin
receptor-mediated processes in adipocytes (13) and showed
that, following its interaction with CD9, GM3 downregu-
lated tumor cell motility and malignancy (14). GM1 was
found to interact with several membrane and cytosolic
proteins (15–18), two of which were characterized as
caveolin-1 (16) and tubulin (17), and that GM3, GM1, and
GD1b interacted with TAG-1 in the plasma membrane
lipid rafts of neuronal cells (19, 20).
Compound 8 (5.8 mmol) was added to a solution of 0.07 M
Ba(OH)2 in dry CH3OH (85 ml) and the mixture was stirred for
24 h at 40°C and filtered under reduced pressure. The precipitate
was washed with ethyl ether and dissolved in water (H2O). The so-
lution was acidified with HCl to pH 3, and extracted twice with
CH2Cl2. The solvent was evaporated under vacuum and the resi-
due purified by flash chromatography and eluted with an 8:2 mix-
ture (v/v) of petroleum ether:ethyl acetate, resulting in compound
9, with a 52% yield. BH3/THF complex (10 ml, 10 mmol, 1.0 M
solution in THF) was added drop wise over 20 min to a solution of
compound 9 (3.7 mmol) in anhydrous THF (20 ml) at Ϫ20°C.
The solution was stirred for 10 min, allowed to warm to room tem-
perature, stirred for 24 h, and quenched with H2O (50 ml) at 0°C.
One gram of solid K2CO3 was added after 10 min and the mixture
was extracted with ethyl ether (3 × 50 ml). The combined organic
layers were dried over Na2SO4 and concentrated under reduced
pressure, resulting in compound 10, with a yield of 81% (24).
To a solution of compound 10 (3 mmol) in dry CH2Cl2 (15 ml)
was added 850 ␮l of triethylamine (Et3N), followed by the drop-
wise addition of methane sulfonyl chloride (MsCl; 4.2 mmol,
This study was designed to show a direct interaction be-
tween C24-LacCer and Lyn. Cells were photolabeled with
tritium-labeled analogs of LacCer having the photoactiva-
ble nitrophenylazide group at the end of the acyl chain.
130
Journal of Lipid Research Volume 56, 2015

Fig. 1. Scheme of the reactions for the synthesis of tritiated and photoactivable LacCer. PentafluorophenolC12NHFMOC or pentafluo-
rophenolC18NHFMOC, hydroxybenzotriazole, Bu3N, DCM/MeOH (a); DDQ, dioxane, 50°C (b); [3H]NaB3H4, MeOH (c); aqueous NH3
(d); Et3N, 4-F-3-NO2-phenilazide, DMF (e). A, n = 1; B, n = 6.
326 ␮l) at 0°C. After allowing it to stand for 30 min, the mixture
was stirred for 4 h at room temperature, diluted in 50 ml CH2Cl2,
and washed three times with 50 ml H2O. The organic layer was
dried over Na2SO4 and evaporated under reduced pressure, re-
sulting in compound 11, with a yield of 93%.
Compound 11 (4.8 mmol) was dissolved in 15 ml anhydrous
dimethylformamide (DMF), to which 15 mmol of NaN3 were
added. The solution was stirred overnight at 45°C, cooled at
room temperature, and concentrated. The crude material was
dissolved in 50 ml CH2Cl2 and washed three times with 50 ml
H2O. The organic phase was dried over Na2SO4 and evaporated
under reduced pressure, resulting in compound 12, with a yield
of 58% (25).
ethyl ether (3 × 50 ml). The combined organic phases were dried
over Na2SO4 and concentrated, resulting in compound 13, at a
yield of 71%.
Compound 13 (2 mmol) was dissolved in 5% CH3COOH in
CH3OH (5 ml) and a catalytic amount of palladium on charcoal
(6 mg) was added in an argon atmosphere. The reaction mixture
was hydrogenated at atmospheric pressure for 20 h. The mixture
was filtered and the catalyst washed with 5% CH3COOH in
CH3OH. The solvent was evaporated, resulting in compound 14,
at a yield of 80%.
Protection and activation of 12-aminododecanoic acid and of
18-amino-octadecanoic acid (compound 14). Protection of (15)
and activation of compound 16 of ␻-amino acids were as previ-
ously described (12).
Compound 12 (2.8 mmol) was dissolved in 0.5 M KOH in
CH3OH (45 ml) and stirred for 48 h. Its pH was adjusted to pH 3
with 1 M HCl, after which it was concentrated and extracted with
LacCer-Lyn interaction through the membrane
131

Fig. 2. Scheme of the reactions for the synthesis of ␻-aminooctadecanoic acid. CH3OH, H2SO4 (a), reflux; Ba(OH)2, MeOH, 40°C (b);
BH3/THF, Ϫ20°C to room temperature (c); MsCl, Et3N, CH2Cl2, 0°C to room temperature (d); NaN3, DMF, 45°C (e); KOH, MeOH (f);
Pd/C, 5% CH3COOH in MeOH (g); 9-fluorenylmethyl chloroformate, 10% Na2CO3 (h); 2-chloro-1-methylpyridinium iodide, pentafluo-
rophenol, Bu3N, CH2Cl2, reflux (i).
N-acylation of lactosylsphingosine with activated ␻-amino
acids, pentafluorophenol-C12NH-FMOC and pentafluorophenol-
Deprotection. Tritiated compound 4A or B (1.7 ␮mol) was
solubilized in 32% aqueous ammonia (2 ml) in a screw-cap flask
for 24 h at room temperature with vigorous stirring. The reaction
mixture was dried and the residue was purified by column chro-
matography with CH2Cl2-MeOH-2 N NH3, 60:35:4 by volume, re-
sulting in compound 5A or 5B, at a yield of 80%.
C18NH-FMOC. pentafluorophenol-C12NH-FMOC
or
pentafluorophenol-C18NH-FMOC, (compound 16, 17 ␮mol),
in anhydrous CH2Cl2 (0.5 ml), 1-hydroxybenzotyriazole (19
␮mol), and tributylamine (Bu3N; 38 ␮mol, 9 ␮l) were added to a
solution of lactosylsphingosine, (compound 16, 16 ␮mol), in an-
hydrous CH3OH (1 ml). After vigorous stirring for 75 min at
room temperature, the reaction mixture was dried and the resi-
due purified by flash chromatography, equilibrated, and eluted
with CHCl3-methanol (MeOH)-H2O, 60:25:4 by volume, result-
ing in compound 2A or 2B, each at a yield of 80%.
Azide labeling. This final reaction was conducted in a dark
room. Radioactive compound 5A or 5B (10 ␮mol/ml) was dissolved
in anhydrous DMF. An equimolar quantity of Et3N and a 2-fold mo-
lar quantity of 4-F-3-NO2-phenylazide, dissolved in ethanol, were
added and the mixture was stirred overnight at 80°C. The reaction
mixture was concentrated and compound 6A or compound 6B was
purified by flash chromatography with CH2Cl2-MeOH-2-propanol-
H2O, 70:10:20:3 by volume. Fractions containing the homoge-
neous tritiated and photoactivable LacCer were immediately
solubilized in MeOH and stored at 4°C in a dark glass bottle.
Oxidation of 2 at the 3-position of sphingosine. Compound 2A
or 2B (12 ␮mol) was suspended in 3% 2,3-dichloro-5,6-dicyano-1,
4-benzoquinone (DDQ) in dioxane (10 ml) and incubated at 50°C
for 40 h with vigorous stirring in a screw-capped tube. The reaction
mixture was evaporated to dryness under vacuum, and excess
DDQ was eliminated by flash chromatography with acetone-
CH2Cl2-CH3OH, 47:2:1 by volume. Fractions containing com-
pounds 3A or 3B were concentrated and the residue was purified
by flash chromatography with CHCl3-MeOH-2-propanol-H2O,
70:5:25:3 by volume, resulting in purifying pure compounds at
yields of 80%.
Treatment of cells with [3H]LacCer-(N3)
All procedures before exposure to UV light were performed
under red safelight (11–13, 15, 18–20, 26–28). To load D-HL-60
cells with [3H]LacCer-(N3), 10 ml aliquots of D-HL-60 cells (2 × 107
cells/ml) in Dulbecco’s PBS were incubated with 0.25 ␮g LacCer
and 0.25 ␮g [3H]LacCer-(N3) (final concentrations 0.5 ␮g/ml)
(7, 12) for 30 min at 20°C with gentle shaking (30 strokes/min).
[3H]LacCer-(N3) was diluted with LacCer to reduce self-quenching
after illumination. After incubation, the cells were washed once
with 10 ml PBS containing 0.1% BSA to remove unincorporated
LacCer and membrane adherent aggregates of LacCer (7). The
cells were washed twice with 10 ml of cold PBS and maintained
in 15 ml of cold PBS. Cells were illuminated for 45 min under UV
light (␭ = 360 nm) on ice.
Tritiation with [3H]NaBH4. Solid [3H]NaBH4 (50 mCi) was
added to compound 3A or 3B (1.9 ␮mol) dissolved in MeOH
(1 ml) and incubated at room temperature for 48 h with vigorous
stirring. The reaction mixture was dried, and the tritiated com-
pound 4A or 4B with a 2S,3R configuration was purified by flash
chromatography with CH2Cl2-MeOH-2-propanol-H2O, 70:10:20:3
by volume.
132
Journal of Lipid Research Volume 56, 2015

After photoactivation, the cells were suspended in lysis buffer
[1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM
EDTA, 1 mM diisopropyl fluorophosphate (DFP), 1 mM Na3VO4,
1 mM PMSF, and 1/20 (v/v) cOmplete (Roche Diagnostics,
Mannheim, Germany)] at 4°C for 20 min. Cell lysates were gently
Dounce homogenized (70 strokes) and centrifuged (1,300 rpm
for 5 min) to remove nuclei and cellular debris. The post nuclear
supernatant (PNS) was removed and transferred to new tubes.
After loading with [3H]LacCer-(N3), cell viability was assessed
by trypan blue exclusion (29, 30). Cell viabilities before and after
loading cells were >90%.
C24-[3H]LacCer-(N3), were diluted with 10 vol of immunoprecipi-
tation buffer [50 mM HEPES (pH 7.5), 1% Triton X-100, 150 mM
NaCl, 2 mM Na3VO4, 10 mM NaF, with 1/20 cOmplete] and
precleared by incubation with 30 ␮l rat anti-mouse IgM/IgG IgG-
bound Dynabeads (Invitrogen) for 1 h at 4°C. The supernatants
were subsequently incubated overnight at 4°C with 5 ␮g anti-
LacCer IgM Huly-m13 or anti-Lyn IgG, followed by incubation
for 4 h at 4°C with 30 ␮l rat anti-mouse IgM/IgG IgG-bound
Dynabeads; as controls, the supernatants were incubated with
normal mouse IgM or IgG. The immunoprecipitates were washed
three times with immunoprecipitation buffer, denatured under
nonreducing conditions, separated on 7.5% polyacrylamide gels,
and transferred onto PVDF membranes.
Phagocytosis assays
[3H]LacCer-(N3) loaded D-HL-60 cells were incubated with
Alexa 647-conjugated non-opsonized zymosan at a concentration
of 10 particles per cell for 45 min at 37°C in DMEM/F12. After
incubation, the cells were washed with ice-cold PBS and fixed
with 2% paraformaldehyde in PBS for 20 min on ice. At least 200
cells in 10 fields per sample were observed using a Leica TCS-SP2
confocal microscope equipped with a Plan-Apochromat ×100 oil
differential interference contrast objective (Leica Microsystems,
Wetzlar, Germany). Phagocytic index was defined as the percent
of cells positive for ingestion multiplied by the average number
of phagocytosed particles per cell (7).
In some experiments, the immunoprecipitated samples were
dissolved in 1.0 ml RIPA buffer [30 mM HEPES (pH 7.4), 150 mM
NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,
5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 1 mM PMSF, 1/20
(v/v) CompleteTM] and immunoprecipitated as above using
RIPA buffer. The presence of Lyn protein was assessed by immu-
noblotting with specific antibody, followed by reaction with sec-
ondary HRP-conjugated antibody (7, 12, 13, 32).
Analysis of protein patterns
The concentrated DRM fractions prepared from illuminated
C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3) loaded cells were
denatured under nonreducing conditions, separated on 7.5%
polyacrylamide gels, and transferred onto PVDF membranes.
Proteins cross-linked to tritium-labeled LacCer derivative were
detected by digital autoradiography. The presence of Lyn and
G␣i was assessed by immunoblotting with specific antibodies, fol-
lowed by reaction with secondary HRP-conjugated antibody (11,
12, 15, 18, 19, 27, 30, 33).
Determination of Lyn phosphorylation
All procedures before illumination were performed under red
safelight. Culture dishes (10 cm) were coated with 5 ml of 10 ␮g/ml
anti-LacCer mAb T5A7 or normal IgM solutions as described (5).
To each dish were added 5 ml of 2.5 × 106 [3H]LacCer-(N3)
loaded D-HL-60 cells per ml PBS containing 1 mM MgSO4 and
1 mM CaCl2, and the dishes were incubated for 5 min at 37°C.
Reactions were terminated by placing on ice. The cells were illu-
minated for 45 min under a UV lamp to induce photo-activation.
The cells were washed and solubilized, the lysates were cleared
by centrifugation and the supernatants were subjected to 8.5%
SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF)
membranes. To measure Lyn phosphorylation, the blots were in-
cubated with anti-phospho-Lyn IgG and then with HRP-conjugated
secondary antibody, followed by detection of phosphorylated
Lyn using SuperSignalTM reagent (Pierce Chemical Company,
Rockford, IL). The membranes were subsequently stripped of
antibody by incubation with stripping buffer [62.5 mM Tris-HCl
(pH 6.8), 100 mM ␤-mercaptoethanol, 2% SDS] for 30 min at
55°C, and then reprobed with anti-Lyn IgG.
Other experimental procedures
1H and 13C NMR spectra were recorded with a Bruker
AVANCE-500 spectrometer at a sample temperature of 298 K.
NMR spectra were recorded in CDCl3 or CD3OD and calibrated
using the TMS signal as internal reference.
Mass spectrometric analyses were performed in positive or
negative ESI-MS. MS spectra were recorded on a Hewlett-Packard
HP-5988-A or a Thermo Quest Finnigan LCQ™ DECA ion trap
mass spectrometer, equipped with a Finnigan ESI interface; data
were processed by Finnigan Xcalibur software system.
All reactions were monitored by TLC on silica gel 60 F-254
plates (Merck).
Flash column chromatography was performed on silica gel 60
(230–400 mesh, Merck).
Radioactivity associated with cells and cell fractions was deter-
mined by liquid scintillation counting. Digital autoradiography
of the PVDF membranes was performed with a Beta-Imager 2000
(Biospace, Paris).
Preparation of detergent-resistant membrane fractions
by sucrose gradient centrifugation
PNS fractions obtained after illumination of [3H]LacCer-(N3)
loaded cells were mixed with an equal volume of 85% of sucrose
(w/v) in buffer A [10 mM Tris buffer HCl (pH 7.5), 150 mM NaCl,
5 mM EDTA, 1 mM Na3VO4], placed at the bottom of a discontinu-
ous sucrose concentration gradient (30–5%) in the same buffer,
and centrifuged for 17 h at 200,000 g at 4°C. After ultracentrifuga-
tion, 12 fractions were collected, starting at the top of the tube. The
light-scattering band, corresponding to detergent-resistant mem-
brane (DRM), was located at the interface between 5% and 30%
sucrose and corresponded to fraction 5. The entire procedure was
performed at 0–4°C in ice immersion (7, 29, 31). In some experi-
ments, the DRM fractions were diluted with 10 vol of buffer A, and
then concentrated at 200,000 g for 1 h at 4°C. The resulting pellets
were subjected to SDS-PAGE and Western blotting analysis.
RESULTS
Preparation of C18-[3H]LacCer-(N3) and C24-[3H]
LacCer-(N3)
The two LacCer species, C18-[3H]LacCer-(N3) and C24-
[3H]LacCer-(N3), were prepared using the strategy previ-
ously developed for the synthesis of photoactivable
gangliosides, modified to introduce tritium at position
3 of sphingosine. A specific strategy was also developed
to synthesize ␻-amino-octadecanoic acid. The reaction
schemes are shown in Figs. 1 and 2.
Immunoprecipitation experiments
The lysates, equivalent to 1.2 × 107 cells, or DRM fractions
from D-HL-60 cells loaded with C18-[3H]LacCer-(N3) and
LacCer-Lyn interaction through the membrane
133

All together, the yield of the GSL synthetic process was
around 50%, which was very high considering the final
specific radioactivity of 1.5 Ci/mmol. Figure 3 shows the
analytical controls as MS and NMR spectra of the synthe-
sized 18-amino-octadecanoic acid (Fig. 2), confirming its
structure. Figure 3 also shows the TLC of the two final
compounds, C18-[3H]LacCer-(N3) and C24-[3H]LacCer-
(N3), showing that both were 98% homogeneous.
D-HL-60 cell viability after administration of C18-[3H]
LacCer-(N3) and C24-[3H]LacCer-(N3)
Cells were administered the photoactivable species of
LacCer together with the natural species, for a total of
0.5 ␮g/ml of cells. Photoactivable LacCer was diluted with
the natural compound to reduce self-quenching at the
moment of illumination. Trypan blue assays showed that
the viability of untreated cells was 96.33 1.6%; when
loaded with C18-[3H]LacCer-(N3) and C24-[3H]LacCer-
(N3), they had a viability of 98.33 0.3% and 96.00 1.5%,
respectively.
Effects of C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3)
on the phagocytosis of microorganisms by D-HL-60 cells
LacCer has been shown to be present in the Triton-
X-100 insoluble membrane fractions of neutrophils and
D-HL-60 cells (5, 7). After administration of exogenous
C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), the cells
were illuminated under UV light, harvested, and lysed in
lysis buffer containing Triton X-100. The lysates were
loaded onto discontinuous sucrose gradients and ultra-
centrifuged; 12 fractions were collected from the top of
each tube and their amounts of radioactivity analyzed.
Figure 4 shows that, following loading with both C18-[3H]
LacCer-(N3) and C24-[3H]LacCer-(N3), part of the radio-
activity was associated with the low density fraction (DRM),
corresponding to fraction 5, which is resistant to detergent
solubilization. Radioactivity associated with high density
fractions 9–12 was due to the photoactivable LacCer mol-
ecules peripherally associated with membrane components,
but not correctly inserted into the membrane bilayer, or to
their catabolites formed after internalization and degrada-
tion (12). Thus, this radioactivity does not reflect a physi-
ological interaction.
To determine whether the distribution of radioactivity
was dependent on the process of illumination, gradient
fractions were also prepared from nonilluminated cells.
Figure 4 shows that the illumination process did not sig-
nificantly alter the distribution of radioactivity.
To verify whether the two LacCer analogs maintain the
biological properties of the corresponding natural LacCer
species, we assessed the phagocytosis activity of D-HL-60
cells loaded with C18-[3H]LacCer-(N3) and C24-[3H]LacCer-
(N3). The first step of the signaling process ultimately lead-
ing to phagocytosis is represented by Lyn phosphorylation.
Thus, D-HL-60 cells loaded with [3H]LacCer-(N3), but not
illuminated, were incubated in the presence of anti-
LacCer T5A7 (stimulated cells) or normal IgM (resting cells)
for 5 min at 37°C. Cell lysates were immunoprecipitated
with mouse anti-Lyn IgG or normal mouse IgG, and the
Fig. 3. Analytical controls. 1: ESI-MS (positive-ion mode): m/z =
300 [M+H]+; 599 [2M+H]+ of the ␻-amino-octadecanoic acid. 2: 1H
NMR (500.13 MHz, MeOD, ppm) 1.18–1.27 [multiplet (m), 26H,
alkyl chain]; 1.46–1.59 (m, 4H, C17, C3); 2.17 [triplet (t), 2H; C2];
2.82 (t, 2H; C18). 3: colorimetric (A) and radioimaging (B) TLC
of standard C18-LacCer; standard C24-LacCer (a), C18-[3H]
LacCer(N3) (b), and C24-[3H]LacCer(N3) (c). TLCs were devel-
oped with the solvent system CH3Cl-CH3OH-2-propanol-H2O,
70:15:15:3 by volume (d).
134
Journal of Lipid Research Volume 56, 2015

Fig. 4. Distribution of radioactivity in sucrose gradient fractions of D-HL-60 cells after incubation with photoactivable and radioactive Lac-
Cer derivatives. Cells were incubated in the presence of a 0.25 ␮g LacCer plus 0.25 ␮g [3H]LacCer-(N3) as described in the Materials and
Methods section. Cells were (A) or were not (B) illuminated for 45 min under UV light to allow cross-linking between LacCer and cellular
components. The cells were subsequently subjected to sucrose gradient ultracentrifugation to prepare plasma membrane microdomains
(DRM fraction). Twelve fractions were collected from the top of the tube, with fraction 5 corresponding to the DRM fractions and fractions
9–12 corresponding to the high density (HD) fractions. The radioactivity associated with each fraction was determined by liquid scintillation
counting. Data are expressed as percentages of total radioactivity. Data are the mean SD of three independent experiments.
immunoprecipitates were separated by SDS-PAGE and
blotted onto PVDF membranes, which were probed with
rabbit anti-pY369-Lyn IgG (P-Lyn) and rabbit anti-Lyn
IgG. Figure 5 clearly shows that Lyn was phosphorylated
only in cells loaded with C24-[3H]LacCer-(N3), whereas
Fig. 6 shows that D-HL-60 cells loaded with C24-[3H]Lac-
Cer-(N3), but not illuminated, maintained the property of
cells loaded with natural C24-LacCer and were capable of
phagocytosing microorganisms. In contrast, phagocytosis
did not occur when the cells were loaded with C18-[3H]
LacCer-(N3), similar to findings with natural C19-LacCer
(7). These results confirm that LacCer requires a long
fatty acyl chain to activate Lyn and induce the phagocytic
process and suggest that the photoactivable LacCer deriva-
tive is suitable for studying the properties of the natural
compound.
by anti-Lyn and anti-LacCer antibodies (5, 7). To determine
whether C24-[3H]LacCer-(N3) has the same properties as
native C24-LacCer, we attempted immunoprecipitation with
anti-LacCer and anti-Lyn antibodies. We first performed
immunoprecipitation experiments using anti-LacCer anti-
body on PNS prepared from D-HL-60 cells loaded with
C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), followed
by SDS-PAGE of the immunoprecipitates and immunob-
lotting with anti-Lyn antibody. Figure 7 shows that similar
amounts of radioactivity were present in the immunopre-
cipitates from D-HL-60 cells loaded with the C24 and C18
derivatives (Fig. 7A). However, Lyn was present in immu-
noprecipitates from cells loaded with C24-[3H]LacCer-
(N3), but not from cells loaded with C18-[3H]LacCer-(N3)
(Fig. 7B). These results indicate that only LacCer contain-
ing very long fatty acids is present in the same membrane
domain as Lyn.
Coimmunoprecipitation of C24-[3H]LacCer-(N3) and Lyn
To confirm these findings, we performed immunopre-
cipitation from DRM fractions with anti-Lyn and under
disaggregating conditions. Figure 8 shows that Lyn was
C24-LacCer and Lyn have been shown to belong to the
same membrane domain, with both immunoprecipitated
LacCer-Lyn interaction through the membrane
135

Fig. 5. Phosphorylation of Lyn after [3H]LacCer-
(N3) loading of D-HL-60 cells. D-HL-60 cells loaded
with 0.25 ␮g LacCer plus 0.25 ␮g [3H]LacCer-(N3)
for 30 min at 20°C, without illumination, were incu-
bated in the presence of anti-LacCer T5A7 (stimu-
lated cells) or normal IgM (resting cells) for 5 min
at 37°C (8). The cell lysates were immunoprecipi-
tated with mouse anti-Lyn IgG or normal mouse
IgG. The immunoprecipitates were separated by
SDS-PAGE and blotted onto PVDF membranes,
which were probed with rabbit anti-pY369-Lyn IgG
(p-Lyn). To evaluate the recovery of Lyn, the blotted
membrane was probed with rabbit anti-Lyn IgG. A:
Blots shown are representative of three indepen-
dent experiments. B: The ratio of the band intensity
of phosphorylated Lyn to total Lyn for each lane was
calculated. Data are presented as ratios relative to
the phosphorylation rate of the resting cells loaded
with C18-[3H]LacCer-(N3) (without anti-LacCer IgM),
and are expressed as the mean SD of three inde-
pendent experiments. *P < 0.05.
immunoprecipitated from DRM fractions prepared from
cells loaded with both C18-[3H]LacCer-(N3) and C24-[3H]
LacCer-(N3), whereas radioactivity was present only in the
latter immunoprecipitate.
Identification of LacCer-protein complexes
LacCer-enriched lipid raft-mediated neutrophil activities
have been shown to involve Src family kinase-, PI-3 kinase-,
and heterotrimeric G-protein-mediated signal transduction
pathways (5, 8). To better identify the radioactive LacCer-
protein complexes, proteins from DRM fractions were con-
centrated, separated by SDS-PAGE, blotted onto PVDF
membranes, and analyzed by digital radioimaging (Fig. 9).
Assays of D-HL-60 cells loaded with C24-[3H]LacCer-(N3)
showed a few radioactive bands within a wide range of mo-
lecular masses, ranging from 20 kDa to 75 kDa, and in-
cluded bands of 41 kDa and 56 kDa (Fig. 9A), suggesting
that specific proteins are present in the membrane environ-
ment of LacCer species containing very long fatty acids. Af-
ter radioimaging, the PVDF membranes were incubated
with antibodies to Lyn and G␣i. These two antibodies bound
specifically to the bands at 56 kDa and 41 kDa, respectively,
suggesting that LacCer may directly interact with Lyn and
G␣i. Indeed, anti-LacCer IgM coimmunoprecipitated Lyn
and G␣i from DRM of D-HL-60 cells loaded with C24-[3H]
LacCer-(N3), but not C18-[3H]LacCer-(N3) (Fig. 9B).
Fig. 6. Phagocytosis index after [3H]LacCer-(N3) loading of
D-HL-60 cells. D-HL-60 cells were loaded with 0.25 ␮g LacCer plus
0.25 ␮g [3H]LacCer-(N3) for 30 min at 20°C, without illumination,
and phagocytosis assays were performed. Each bar shows the mean
of 20 different fields of two independent experiments.
136
Journal of Lipid Research Volume 56, 2015

the hydrolysis of GSLs at the cell surface by GSL hydrolases
are involved in the regulation of apoptosis (39, 40).
In mammals, ceramides are synthesized by a family of
six enzymes, ceramide synthase (CerS)1–6, each of which
uses a relatively restricted subset of fatty acyl-CoAs to
N-acylate the sphingoid long-chain base (41). The levels of
expression of each CerS encoding gene differ among sev-
eral tissues, suggesting that molecular variations and ex-
pression patterns of fatty acid chains of GSLs reflect the
functions of these cells. CerS2 is the predominant CerS of
C24 ceramides (42, 43). CerS2 knockdown mice exhibited
severe hepatopathy, myelin sheath defects, and cerebellar
degeneration. The availability of very long-chain fatty ac-
ids requires ELOVL enzymes in mammals. ELOVL1 shows
high activity toward saturated and monounsaturated C20-
and C22-CoAs, and has been shown to be essential for the
production of C24 fatty acid containing sphingolipids.
ELOVL1 activity is regulated by CerS2. Knockdown of
ELOVL1 or CerS2 markedly reduced Lyn activation in
HeLa cells (44). These results are consistent with our ob-
servation that C24-LacCer molecules are indispensable for
Lyn-coupled LacCer-enriched lipid raft-mediated neutro-
phil functions (7).
LacCer-enriched lipid rafts mediate neutrophil che-
motaxis, phagocytosis, and superoxide generation of neu-
trophils (5, 8, 45). DMSO-differentiated HL-60 cells are
commonly used for analyzing neutrophil functions, in-
cluding migration, phagocytosis of opsonized microor-
ganisms, superoxide generation, and degranulation (5, 8,
46–49). However, those HL-60 cells do not possess LacCer-
mediated neutrophil functions, including migration,
superoxide generation, or phagocytosis, because the pres-
ence of C24-LacCer in plasma membranes is insufficient
to form Lyn-coupled LacCer-enriched lipid rafts (7). In
contrast, C24-LacCer is present in granules of DMSO-
differentiated HL-60 cells, indicating that biosynthesis of
C24-LacCer in itself is normal. It seems, therefore, that the
delivery system of C24-LacCer from Golgi apparatus to
plasma membranes in DMSO-differentiated HL-60 cells is
abnormal. Although it remains unclear why C24-LacCer is
not selectively delivered to plasma membranes from Golgi
apparatus in DMSO-differentiated HL-60 cells, these cells
are a good model to analyze the organization and signal
transduction mechanisms of LacCer-enriched lipid rafts.
LacCer is located on the outer layer of plasma membranes,
whereas Lyn is present at their cytosolic face via myristic/
palmitic acyl chains. It has been suggested that the long
fatty acids of LacCer may interdigitate (50, 51) with the
acyl chains of the cytosolic layer, reducing membrane
thickness (52, 53). This may allow for direct interactions
between LacCer and Lyn, which may be instrumental for
Lyn phosphorylation, considered the starting process nec-
essary for the immunological functions of neutrophils (7).
To date, however, direct interactions between LacCer and
Lyn through the membrane layer have not been observed
experimentally.
Fig. 7. Anti-LacCer immunoprecipitation from PNS. PNS from
D-HL-60 cells loaded with [3H]LacCer-(N3) were immunoprecipi-
tated with mouse anti-LacCer antibody Huly-m13 or normal mouse
IgM. A: The radioactivity associated with the immunoprecipitates
was determined by liquid scintillation counting. Data are expressed
as percentages of total radioactivity. Data are the mean of three dif-
ferent experiments. ***P < 0.0005. B: The immunoprecipitates
were analyzed by SDS-PAGE/immunoblotting using rabbit anti-
Lyn IgG. The immunoblot shown is representative of three differ-
ent experiments.
DISCUSSION
GSLs are components of the outer layer of plasma mem-
branes, with considerable heterogeneity, due to heteroge-
neity of both the oligosaccharide and ceramide moieties.
They are enriched with respect to glycerophospholipids in
limited areas containing a few selected proteins, many of
which are associated with cell signaling (28, 34). GSLs
have been found to modulate cell signaling processes
through specific interactions between their oligosaccha-
ride chains and proteins/glycoproteins in their environ-
ment. In contrast, it is unclear whether ceramides play
specific roles in such processes, although changes in the
structures of both the long-chain base and the fatty acids
have been associated with the differentiation and aging
processes (35–38). In addition, ceramides resulting from
Photoactivable GSLs have been shown to be good tools
to study GSL-protein interactions (13, 14, 16, 17). To ver-
ify the direct interaction between C24-LacCer and Lyn
LacCer-Lyn interaction through the membrane
137

Fig. 8. Anti-Lyn immunoprecipitation from D-HL-60 DRM fractions. DRM fractions from cells loaded with
[3H]LacCer-(N3) were immunoprecipitated with mouse anti-Lyn IgG or normal mouse IgG. A: The immu-
noprecipitates were separated by SDS-PAGE and blotted onto PVDF membranes, which were probed with
rabbit anti-Lyn IgG. The immunoblot shown is representative of three different experiments. B: The radio-
activity associated with the immunoprecipitates was determined by liquid scintillation counting. Data are
expressed as percentages of total radioactivity. Data are the mean of three different experiments. ***P <
0.0005.
through the membrane layers, we synthesized a photoacti-
vable LacCer containing a reactive azide group at the end
of the acyl chain. This chain was of a length similar to that
of C24 fatty acids, as shown by molecular modeling. The
photoactivable C24 fatty acid containing LacCer was la-
beled with tritium at position 3 of sphingosine, yielding
C24-[3H]LacCer-(N3). As a control, we also synthesized
a tritium-labeled photoactivable LacCer containing a
shorter acyl chain, C18-[3H]LacCer-(N3).
with C24-[3H]LacCer-(N3), but not C18-[3H]LacCer-(N3).
These results are similar to those observed when D-HL-60
cells were loaded with natural LacCer molecules contain-
ing very long and short/medium fatty acids, suggesting
that the photoactivable LacCer derivatives mimic the
natural molecules.
After cell loading and illumination, the patterns of
the radioactive proteins were analyzed. Illumination
rapidly transforms the azide to nitrene, a very reactive
group capable of covalently binding to molecules in
their nearest environment. Thus any interaction of
tritium-labeled LacCer derivative with proteins would
yield tritium-labeled LacCer-protein complexes that can
be analyzed by PAGE, followed by radioimaging of the
blotted material.
In this study, we showed that C24-[3H]LacCer-(N3)
covalently binds to Lyn in LacCer-enriched lipid rafts.
D-HL-60 cells were loaded with these two types of [3H]
LacCer-(N3), with percentages of each found to be present
in the DRM fractions prepared from cell lysates. Lyn phos-
phorylation and phagocytosis were observed in cells loaded
138
Journal of Lipid Research Volume 56, 2015

Fig. 9. Identification of the LacCer-protein com-
plexes. A: Proteins cross-linked with C24-[3H]LacCer-
(N3) (lane 1) and C18-[3H]LacCer-(N3) (lane 4)
prepared from DRM fractions were concentrated by
centrifugation (1 h, 200,000 g, 4°C) and separated
by 7.5% SDS-PAGE, blotted onto PVDF membranes,
and visualized by digital autoradiography for 96 h.
The same PVDF membranes were sequentially visu-
alized by Western blotting using rabbit anti-Lyn
(lane 2 and lane 5) and anti-G␣i (lane 3 and lane 6)
IgGs. The results shown are representative of three
independent experiments. B: DRM fractions from
D-HL-60 cells loaded with [3H]LacCer-(N3) were im-
munoprecipitated with anti-LacCer IgM Huly-m13
or normal mouse IgM. The immunoprecipitants
were separated by 7.5% SDS-PAGE, blotted onto
PVDF membrane, and sequentially visualized by
Western blotting using rabbit anti-Lyn and anti-G␣i
IgGs.
The administration of C24-[3H]LacCer-(N3) to the cells
followed by illumination resulted in the association of ra-
dioactivity with the plasma membrane lipid rafts. Most of
the radioactivity was linked to lipids, which was not unex-
pected, because lipids are the main components of lipid
rafts. Some of the radioactivity, however, was linked to pro-
teins, but only a few proteins were labeled, suggesting that
their interactions with LacCer are specific. One of the
cross-linked proteins was identified as Lyn, whereas a sec-
ond protein was stained with anti-G␣i. Because the azide
moiety is at the end of the LacCer fatty acid, which is in-
serted into the outer membrane layer, and Lyn is asso-
ciated with the cytosolic membrane layer by insertion of
its acyl chains, the only mechanism by which a LacCer-
Lyn complex can form is by the acyl chain of LacCer con-
tacting with the acyl chain(s) of Lyn. In contrast, when
C18-[3H]LacCer-(N3) was administered to cells, little radio-
activity was associated with the lipid raft proteins, and a
LacCer-Lyn complex could not be detected.
necessary for transfer of information through the mem-
brane. However, it is difficult to imagine that these interac-
tions are associated with specific hydrophobic moieties.
Rather, LacCer containing C24 fatty acids may promote
the organization of specific lipid rafts necessary to allow
the interaction of several proteins instrumental to the cell
signaling processing. Without C24-LacCer molecules, this
microenvironment would not be available, Lyn would not
be recruited and activated by phosphorylation, and the
signaling process would not be induced.
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