Lipids (2010) 45:593–602
DOI 10.1007/s11745-010-3432-1
ORIGINAL ARTICLE
Lipidomic Analysis of Porcine Olfactory Epithelial Membranes
and Cilia
Simona Lobasso • Patrizia Lopalco • Roberto Angelini
Maristella Baronio • Francesco P. Fanizzi •
Francesco Babudri • Angela Corcelli
•
Received: 7 April 2010 / Accepted: 6 May 2010 / Published online: 29 May 2010
Ó AOCS 2010
Abstract The use of the matrix 9-aminoacridine has been
recently introduced in matrix-assisted laser desorption/
ionization time-of-flight (MALDI-TOF) mass spectrometry
analysis of both anionic and cationic phospholipids. In the
present study, we take advantage of this technique to
analyze the lipids of porcine olfactory mucosa and a
membrane fraction enriched in cilia. Thin-layer chromatography (TLC) and 31P-NMR analyses of the lipid extracts
were also performed in parallel. MALDI-TOF-MS allowed
the identification of lipid classes in the total lipid extract
and individual lipids present in the main TLC bands. The
comparison between the composition of the two lipid
extracts showed that: (1) cardiolipin, present in small
amount in the whole olfactory mucosa lipid extract, was
absent in the extract of membranes enriched in olfactory
cilia, (2) phosphatidylethanolamine species were less
abundant in ciliary than in whole epithelial membranes,
S. Lobasso R. Angelini M. Baronio A. Corcelli (&)
Department of Medical Biochemistry Medical Biology
and Medical Physics, University Aldo Moro, Pl. G. Cesare,
70124 Bari, Italy
e-mail: [email protected]
P. Lopalco
Institute for Microelectronics and Microsystems (IMM),
National Research Council (CNR), Lecce, Italy
F. P. Fanizzi
Department of Biological and Environmental Sciences
and Technologies, University of Salento, Lecce, Italy
F. Babudri
Department of Chemistry, University Aldo Moro, Bari, Italy
A. Corcelli
Institute for Chemical-Physical Processes,
National Research Council (IPCF-CNR), Bari, Italy
(3) sulfoglycosphingolipids were detected in the lipid
extract of ciliary membranes, but not in that of epithelial
membranes. Our results indicate that the lipid pattern of
ciliary membranes is different from that of whole-tissue
membranes and suggest that olfactory receptors require a
specific lipid environment for their functioning.
Keywords Olfactory epithelium Pig Lipids MALDI-TOF-MS TLC 31P-NMR
Abbreviations
AC3
cAMP
Cer
CHOL
CM
DMSO
DTT
Gb5
IBMX
MALDI-TOF-MS
OR
OSN
PtdOH
PtdCho
PtdEtn
p-PtdEtn
PtdIns
PMSF
PtdSer
Ptd2Gro
S-GalCer
Adenylcyclase III
Cyclic-adenosine-monophosphate
Ceramides
Cholesterol
Ciliary membranes
Dimethylsulfoxide
Dithiothreitol
Globopentaosylceramides
Isobutylmethylxanthine
Matrix-assisted laser desorption/
ionization time-of-flight mass
spectrometry
Olfactory receptor
Olfactory sensory neuron
Phosphatidic acid
Phosphatidylcholine
Phosphatidylethanolamine
Plasmenyl-Phosphatidylethanolamine
Phosphatidylinositol
Phenylmethanesulfonyl fluoride
Phosphatidylserine
Cardiolipin
Sulfoglycosphingolipids
123
594
CerPCho
WM
Lipids (2010) 45:593–602
Sphingomyelin
Whole-tissue membranes
Introduction
Inhalation of odors across the surface of the olfactory
epithelium of the animal nose activates the olfactory signaling cascade, which involves the binding of ligands to
receptors localized on primary sensory cells, the olfactory
sensory neurons (OSN). The OSN are bipolar neurons of
the pseudostratified olfactory epithelium, having a thin
sensory axon extending to higher brain regions and a single
dendrite that ends with a knob, from which long fine cilia
protrude, directly projected into the mucous of the olfactory epithelium.
Olfactory cilia are the sites of the sensory transduction
apparatus. The binding of odorants to G-protein-coupled
seven-transmembrane olfactory receptors (OR) activates
the Gaolf subunit of a specific heterotrimeric G-protein
complex, which stimulates the enzyme adenylcyclase III
(AC3) to synthesize the second messenger molecule cyclicadenosine-monophosphate (cAMP). cAMP in turn activates the opening of cyclic-nucleotide-gated ion channels
present on the plasma membrane, generating electrical
signals in the primary sensory axons [1, 2].
Each OSN expresses only one type of OR out of a
repertoire of about 1,000 [3]. Numerous OSN expressing
the same OR are dispersed in the olfactory mucosa, while
their axons converge to form glomeruli in the olfactory
bulb, where a precise distribution or map of odors exists.
It is known that several proteins involved in the sensory
signaling cascade are compartmentalized in specialized
membrane subdomains, called lipid rafts, which are
expected to be spread in the olfactory ciliary membranes
[4, 5].
In the lipidomics era it is surprising to find out that only
a few analyses of the lipids of olfactory mucosa have been
reported in the literature. Old reports described the lipid
composition of plasma membranes isolated from bovine
[6] and rat [7] olfactory mucosa, while no data are available either on pig olfactory mucosa in toto, or on isolated
olfactory cilia.
It would be helpful to have an overview of the specific
set of lipids in the sensory cilia in order to investigate the
possible functions of lipids in signal transduction, adaptation, xenobiotic metabolism and OSN maturation.
The present study provides the first general characterization of the membrane lipids of the neuroepithelial
olfactory mucosa covering pig turbinates and information
on lipids associated with the specialized membrane domain
of pig olfactory cilia.
123
Materials
The nasal cornets of pig were kindly provided immediately
after sacrifice by the slaughterhouse in Ruvo di Puglia (Bari,
Italy). The Cyclic AMP [3H] assay system was purchased
from Amersham (Freiburg, Germany). Odorants (Sigma–
Aldrich, St. Louis, MO) were usually prepared as stock
solutions in ethanol or DMSO. All organic solvents used
were commercially distilled and of the highest available
purity (Sigma–Aldrich). Plates for TLC (Silica gel 60A,
10 9 20 cm, 0.25 mm thick layer), obtained from Merck
(Darmstadt, Germany), were washed twice with chloroform/
methanol (1:1, by vol) and activated at 120 °C before use.
The following lipid standards were obtained from Avanti
Polar Lipids, Inc. (Alabaster, AL): 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14:0 PtdOH), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (14:0
PtdSer), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (16:1 PtdEtn), 1,2-dioleoyl-sn-glycero-3-phospho(10 -myo-inositol) (ammonium salt) (18:1 PtdIns), 10 ,30 -bis
[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (sodium
salt) (14:0 Ptd2Gro), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (14:1 (D9-Cis) PtdCho), 1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PtdCho), 1,2-di-O-phytanylsn-glycero-3-phosphocholine (4ME 16:0 diether PtdCho),
N-palmitoyl-D-erythro-sphingosylphosphorylcholine 16:0
CerPCho (d18:1/16:0). The matrix for MALDI-TOF–MS
(9-aminoacridine hemihydrate) was purchased from Acros
Organics (Morris Plains, MJ).
Methods
Isolation of Membranes Enriched in Olfactory Cilia
Ciliary membranes were detached and isolated from pig
olfactory epithelia as previously described [8, 9]. The pig
olfactory epithelia, situated on both left and right ethmoturbinates, were carefully stripped from the underlying
bone and washed in ice-cold Ringer solution (120 mM
NaCl, 5 mM KCl, 1.6 mM K2HPO4, 1.2 mM MgS04,
25 mM NaHCO3, 7.5 mM glucose, pH 7.4) plus 1 mM
PMSF within 10–20 min of slaughter. All operations were
carried at 0–4 °C. The olfactory cilia were detached using a
calcium shock procedure, raising the calcium concentration
in the Ringer solution up to 10 mM. After gently shaking
(20 min at 4 °C), the deciliated epithelia were removed by
centrifugation (7,000g for 5 min). The supernatant was
collected and the pellet incubated again with the solution
containing 10 mM calcium ion for 20 min. After removing
the deciliated epithelia by centrifugation, the supernatants
containing the detached cilia were combined. Membranes
enriched in olfactory cilia were collected by centrifuging at
Lipids (2010) 45:593–602
27,000g for 15 min, and resuspended in Buffer A (3 mM
MgCl2, 2 mM EDTA, 10 mM Tris/HCl, 1 mM PMSF, pH
7.4) with 5% glycerol. Ciliary membranes were apportioned into small volumes and saved at -80 °C. The protein concentration was determined by the Bradford method
[10], with bovine serum albumin as standard.
Whole-Tissue Membrane Isolation
Pig olfactory epithelia, carefully dissected as described in
the previous paragraph, were suspended in 5–7 volumes of
hypotonic Ringer solution (Ringer without NaCl) and
homogenized on ice with a Potter homogenizer. Cartilage
fragments and large epithelia pieces were removed by low
speed centrifugation followed by filtration. The filtrate was
then centrifuged twice at 1,500g for 10 min and the pigmented pellet removed. The final supernatant was centrifuged at 27,000g for 20 min to generate a light yellow
pellet. This pellet was suspended in 5% glycerol-containing
Buffer A and stored at -80 °C.
Adenylcyclase Assay
Adenylcyclase activity was assayed according to a modified version of the method described previously [11, 12].
All assays were carried out at 37 °C. Briefly 70 ll of cilia
suspension (50–100 lg protein/ml) was mixed with 360 ll
of stimulating buffer containing 200 mM NaCl, 10 mM
EGTA, 50 mM MOPS, 2.5 mM MgCl2, 1 mM DTT,
0.05% sodium cholate, 1 mM ATP, 20 lM GTP, 1 mM
IBMX buffered at pH 7.4, with or without a stimulant
(200 lM odorants or 5 lM forskolin). The incubation of
cilia with the stimulating buffer (15 min at 37 °C) was
stopped by adding 350 ll of ice cold 10% perchloric acid.
Then samples were centrifuged at 2,500g for 5 min at 4 °C;
400 ll of the supernatant was added to 100 ll of 10 mM
EDTA pH 7.0. The samples were then neutralized by
adding 500 ll of a mixture containing 1,1,2-trichlorotrifluoroethane and tri-n-octylamine, mixed and then three
phases were obtained by centrifugation; the upper phase
contained the water soluble components and was used to
estimate the amount of cAMP produced during the incubation of cilia with substrates. The cAMP was determined
by radioimmunoassay using an Amersham kit.
Lipid Extraction
Total lipids were extracted from membranes using the
Bligh and Dyer method [13] in the presence of 0.01% DTT
as antioxidant. 6 ml of methanol/chloroform (2:1, by vol)
was added to a 1.6 ml membrane suspension (about 6 mg
proteins). The mixture was gently shaken for 15 min and
then centrifuged to collect the supernatant. The residue
595
pellet was re-extracted by adding 7.6 ml of methanol/
chloroform/water (2:1:0.8, by vol). The mixture was again
shaken for 15 min and centrifuged. Then 4 ml each of
chloroform and 0.2 M KCl were added to the combined
supernatant extracts to obtain a two-phase system, chloroform and methanol/water (1:0.9, by vol). After complete
phase separation, the lipid-containing chloroform phase
was brought to dryness under argon; dried lipids were
weighed, suspended in a small chloroform volume and
saved at -20 °C.
Thin-Layer Chromatography
Total lipid extracts were analyzed by TLC on silica gel
plates and lipids were eluted with Solvent A, chloroform/
methanol/acetic acid/water (85:15:10:3.5, by vol). For
2D-analysis, the plates were developed using Solvent B,
chloroform/methanol/ammonium hydroxide (65:25:5, by
vol), in the first dimension, and Solvent A in the second
dimension. The solvent used for separation of neutral lipids
on 1D-TLC was Solvent C, hexane/ethyl ether/acetic acid
(70:30:1, by vol). Individual phospholipids were identified
by reference to authentic lipid standards (Sigma–Aldrich).
Lipids were detected by spraying plates with 5% sulfuric
acid, followed by charring at 120 °C. Additional confirmation of the identity of lipids was obtained using (a)
molybdenum blue reagent (Sigma–Aldrich), specific for
phospholipids, (b) 0.5% a-naphthol in methanol/water (1:1,
by vol), specific for glycolipids, (c) ninhydrine 0.25% in
acetone/lutidine (9:1, by vol), for free amino groups [14].
To analyze in detail the various lipid components of the
extracts, bands present on 1D-TLC, developed in Solvent
A, were scraped and lipids extracted from silica, as previously described [14]; then lipid bands were analyzed by
mass spectrometry.
MALDI-TOF Mass Spectrometry
Lipid analysis was performed as previously described [15].
Briefly total lipid extracts (10 mg/ml; dissolved in chloroform/methanol (1:1, by vol)) were diluted from 20 to
200 ll with isopropanol/acetonitrile (60:40, by vol). After
mixing 10 ll of diluted sample with 10 ll of 9-aminoacridine (10 mg/ml; dissolved in isopropanol/acetonitrile
(60:40, by vol)), 0.3 ll of the mixture was spotted on the
instrument plate. The same procedure was followed to
analyze the lipid standards (1 mg/ml). MS analysis was
performed on a Bruker Microflex spectrometer (Bruker
Daltonics, Bremen, Germany). Mass spectra were acquired
in the positive and negative mode by averaging 600 consecutive laser shots (50 shots per subspectra). Synthetic
lipid standards (Avanti Polar Lipids) were used as external
standards for calibration.
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NMR Spectroscopy
31
P-NMR analysis of phospholipids present in the total
lipid extract was performed by following the previously
described method [16, 17]. The method is based on the use
of a methanol reagent containing D2O and a dissolved
EDTA salt, prepared as follows. The cesium salt of EDTA
was prepared by titrating a 0.2 M suspension of EDTA free
acid with CsOH to a pH of 6.0, at which point free EDTA
was in solution; EDTA salt solutions were evaporated to
dryness on a freeze-dry apparatus, dissolved in a minimum
volume of D2O to exchange labile 1H for 2D, dried a
second time and dissolved in D2O to a concentration of
0.2 M. The final methanol reagent was prepared by dissolving 1 ml of D2O-EDTA solution in 4 ml of methanol.
The use of D2O is solely in order to provide a deuterium
reference signal for magnetic resonance field-frequency
stabilization; it is not essential for signal narrowing.
Between 1 and 5 mg of phospholipid standards and 12 mg
of total lipid extract were dissolved in 0.8 ml of deuterated
chloroform. To this solution 0.4 ml of methanol reagent
(containing Cs/EDTA) was added, and the mixture stirred
gently. Two liquid phases were obtained, a larger chloroform phase and a smaller water phase. By using a Pasteur
pipette, the sample was placed in an NMR test tube, where
it separated within 1 min. The sample tube turbine was
adjusted so that only the chloroform phase was detected by
the NMR spectrometer’s receiver coil. Magnetic field stabilization was obtained through the deuterium resonance of
deuterated chloroform. Unless otherwise specified, samples
were analyzed with proton broad-band decoupling to
eliminate 1H-31P multiplets. Under these conditions each
spectral resonance corresponded to single phosphorus. 31P
chemical shifts were relative to 85% H3PO4 as an external
standard. Samples were analyzed using a Bruker DRX500
Avance instrument (Bruker Daltonics, Bremen, Germany).
Results
Whole-tissue membranes (WM) of neuroepithelial cells
were isolated from pig olfactory epithelium and total lipids
were extracted to be analyzed; in addition to the WM, a
membrane fraction enriched in olfactory cilia (CM) was
isolated by following the so-called calcium-shock method
[8, 9], briefly described in Fig. 1.
Ciliary membrane enrichment was estimated by assay of
odor-stimulated AC3 activity (i.e. an activity marker for
olfactory cilia); Table 1 reports the specific AC3 activity
both in membranes enriched in olfactory cilia and in
the whole-tissue membranes, in the absence and in the
presence of stimulants. Basal, forskolin-stimulated and
odor-stimulated AC3 activity was observed in both the
123
Fig. 1 Calcium-shock scheme for isolating membranes enriched in
olfactory cilia. The pig olfactory epithelia were suspended in ice-cold
Ringer solution containing 10 mM CaCl2 (twice). To separate tissue
fragments, fractions were centrifuged at low speed (7,000g for
5 min). To collect membranes enriched in olfactory cilia, the
combined supernatants were centrifuged at high speed (27,000g for
15 min). The final membrane pellet was resuspended in Buffer A/5%
glycerol and stored at -80 °C
membrane preparations, but it is evident that forskolin- and
odor-stimulated activities were higher in the membrane
fraction enriched in cilia.
Total lipids were extracted from the two different
membrane preparations isolated from epithelial cells of pig
nose; the lipid/protein ratio was about 1.2 in both membrane fractions. Figure 2 shows the two-dimensional TLC
analyses of the total lipid extracts of CM (a) and WM (b).
Lipid band identification was performed by comparison
with authentic standards (St, in Fig. 2c). The two lipid
profiles were similar; five main bands arose from the separation of polar lipid components during the chromatographic run, while neutral lipid bands could be seen close
to the solvent front. Polar lipid bands corresponded to the
following lipid classes (in Rf order): sphingomyelin
(CerPCho) (as a doublet), phosphatidylinositol (PtdIns),
phosphatidylcholine (PtdCho), phosphatidylserine (PtdSer), and phosphatidylethanolamine (PtdEtn); cardiolipin
(Ptd2Gro) was a minor lipid component present in the lipid
extract of WM, while it was absent in the lipid extract of
CM. Another difference between the two TLC plates in
Fig. 2a, b was in the intensity of PtdEtn spot, which was
more intense in whole than ciliary membranes. Glycosphingolipids (cerebrosides, gangliosides, sulfatides), relatively abundant components of nervous tissue cells, could
Lipids (2010) 45:593–602
597
Table 1 Adenylcyclase activity in isolated membranes
Basal activity ± SEM
Membranes enriched in cilia
Whole-tissue membranes
Forskolin-stimulated activity ± SEM
Odor-stimulated activity ± SEM
197.7 ± 25.3
850.5 ± 39.4
480.1 ± 26.6
60.9 ± 18.6
280.3 ± 13.2
150.5 ± 10.2
Cyclase assay was carried out with 5 lM forskolin or with an odor mixture containing 100 lM each of eugenol and citralva, as described in
‘‘Methods’’. The specific activity is reported as pmol cAMP/mg/min. The values are averages of three separate experiments. SEM refers to
standard error of the mean
Fig. 2 2D-TLC of the lipid
extracts of membranes isolated
from pig olfactory epithelium.
Total lipids were extracted from
the membrane preparations
isolated from epithelial cells of
pig nose and from membranes
enriched in olfactory cilia, as
described in ‘‘Methods’’. Eighty
micrograms of the lipid extract
of whole-tissue (WM) and
enriched in olfactory cilia (CM)
membranes were loaded in the
TLC plates (a) and (b),
respectively. The following pair
of solvents was used: Solvent B
chloroform/methanol/
ammonium hydroxide (65:25:5,
by vol), and then Solvent A
chloroform/methanol/acetic
acid/water (85:15:10:3.5, by
vol). Total lipids were detected
by spraying with 5% sulfuric
acid and charring at 120 °C. On
the TLC plate in (c) eight
micrograms of each lipid
standard were loaded. S-GalCer,
galactocerebroside sulfate;
Ptd2Gro, cardiolipin; CHOL,
cholesterol; PtdCho,
phosphatidylcholine; PtdEtn,
phosphatidylethanolamine;
PtdIns, phosphatidylinositol;
PtdSer, phosphatidylserine;
CerPCho, sphingomyelin
be not detected by this technique either in whole or in
ciliary membranes. Finally it was difficult to compare the
cholesterol (CHOL) spots in WM and CM, because they
were too close to the solvent front overlapping with other
neutral lipid components, such as fatty acids and diacylglycerols. All together the TLC data in Fig. 2 document
that our preparation of ciliary membranes was devoid of
mitochondrial membranes and is characterized by a lower
PtdEtn content than the whole membranes.
The difference in the PtdEtn content was also evident in
the 1D-TLC lipid profiles (Fig. 3). To further investigate
on the CHOL content of WM and CM, we analyzed their
lipid extracts by 1D-TLC using a solvent for neutral lipids
(as described in ‘‘Methods’’). The results revealed that
cholesterol percentage did not differ between the two lipid
extracts and amounted to about 10% (Fig. 4).
To gain detailed information on the lipid classes present
in pig olfactory mucosal cells, the total lipid extract of
whole-tissue membranes was analyzed by MALDI-TOFMS, using the novel matrix 9-aminoacridine allowing a fast
reliable analysis of both zwitterionic and anionic lipid
species [15]. Main lipid bands isolated from preparative
1D-TLC were also analyzed by MALDI-MS. Furthermore
ESI-MS analysis of the lipid extract was performed to
support the identification of lipid classes by MALDI-TOFMS (not shown).
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Lipids (2010) 45:593–602
Fig. 3 1D-TLC lipid profiles of whole-tissue (WM) and enriched in
olfactory cilia (CM) membranes. Total lipids (60 micrograms) were
detected by charring. Solvent A was used
Fig. 4 Cholesterol content of whole-tissue (WM) and enriched in
olfactory cilia (CM) membranes. Ten micrograms of each lipid
extract was loaded on the TLC plate. The following solvent was used:
Solvent C, hexane/ethyl ether/acetic acid (70:30:1, by vol). Only the
CHOL bands are shown (note that with Solvent C polar lipids do not
separate and are all together at the sample deposition line). The
quantitative analysis of CHOL content was performed by videodensitometry (ImageJ software), using standard CHOL loaded on the
same plate to construct the calibration curve
The mass spectrum acquired in the negative mode
(Fig. 5a) showed two main peaks at m/z 885.8 and 788.8,
corresponding to the molecular ions [M - H]-of PtdIns
38:4 and PtdSer 36:2, respectively. The minor peaks at m/z
750.6, 766.7 and 810.7 corresponded to the molecular ions
[M - H]- of plasmenyl-PtdEtn, PtdEtn and PtdSer (all
38:4 species), respectively. Furthermore the peaks at m/z
838.7 and 857.3 were attributed to a PtdSer 40:4 and PtdIns
36:4. Two small peaks were present in the high m/z range;
the peak at m/z 1448.3 was attributed to the molecular ions
[M - H]- of a cardiolipin, having four linoleic acid (18:2)
123
Fig. 5 MALDI-TOF mass spectrum profiles of the lipid extract of pig
epithelial membranes in the negative (a) and positive (b) mode. The
insets enlarge the two regions m/z 900–1,000 and 1,400–1,500 and
show the comparison of the lipid profile of membranes isolated from
the whole epithelium (lower line) with that of membranes enriched in
olfactory cilia (upper line). PtdOh, phosphatidic acid; p-PtdEtn,
plasmenyl-phosphatidylethanolamine; S-GalCer, galactosylceramide
sulfated; Gb5, globopentaosylceramides
chains, while the peak at m/z 1475.8 was not assigned. Of
course the apparent amounts of the various lipids in the
MALDI profiles depend on the lipid individual tendency to
ionization together with its abundance in the extracts.
MALDI-TOF-MS analysis also allowed the direct
identification of minor lipid components of olfactory neuroepithelial cells, such as sulfoglycosphingolipids (i.e.
sulfatides). A detailed description of the advantages of
MALDI-TOF in the analysis of sulfatides has been recently
reported [18].
Lipids (2010) 45:593–602
599
were similar to those of whole-tissue membranes. Some
differences were found in the spectrum acquired in the
negative mode, in the intervals of m/z values 900–1,000
and 1,200–1,600. These regions of the MALDI-TOF mass
spectrum are reported in the insets of Fig. 5a, in order to
allow direct comparison of MALDI-TOF-MS lipid profiles
of the two different lipid extracts. It can be seen that in the
mass spectrum of CM lipid extract: (a) cardiolipin was
absent, in agreement with previous TLC data (see Fig. 2a);
(b) additional peaks were present between m/z 930–960
suggesting that the ciliary membranes contained other
sulfoglycosphingolipids, besides the S-GalCer at m/z 906
(see inset in Fig. 5a).
This last finding arises from the particularly high sensitivity of MALDI-MS in the detection of sulfoglycosphingolipids in the tissue lipid extracts; the cluster centered
at m/z 934 indicates, on qualitative basis, the presence of an
enrichment of long-chain sulfoglycosphingolipids (S-GalCer 26:0) in the specialized membrane of cilia.
Tables 2 and 3 report the main phospholipid and glycolipid classes, resulting from the MALDI-TOF-MS analysis of the total lipid extracts and individual TLC bands.
Like lipids of nervous tissue, polar lipids of the olfactory
mucosa contain mainly arachidonic (20:4) and stearic
(18:0) fatty acids.
Finally, in order to check for quantitative differences in
lipid composition between the whole-tissue and ciliary
membranes, the total lipid extracts of both the membrane
preparations were analyzed by 31P-NMR spectroscopy. In
order to assign NMR peaks to the lipid components in the
extracts, authentic standard phospholipids were analyzed
under the same experimental conditions and their chemical
shifts are reported in Table 4.
The peak at m/z 906.6 in the inset of the mass spectrum
of Fig. 5a can be attributed to a sulfoglycosphingolipid,
precisely to the sulfated galactosylceramide (S-GalCer),
consisting of a C24:0 hydroxy-fatty acid plus the sphingoid
4-sphingenine (d18:1).
The mass spectrum acquired in the positive mode
(Fig. 5b) was dominated by PtdCho species: the peaks at
m/z 734.3, 760.4, 786.6 and 810.5 corresponded to the
molecular ions [M ? H]? of PtdCho 32:0, 34:1, 36:2 and
38:4, respectively; the minor peak at m/z 703.4 can be
attributed to the molecular ion [M ? H]? of the sphingolipid CerPCho 16:0.
Furthermore the peaks at higher m/z suggested the presence of other complex glycosphingolipids, such as
gangliosides. The peaks between m/z 1,400 and 1,600
were attributed to globopentaosylceramides (Gb5), which
give strong [M ? Na]? ions, corresponding to glycosphingolipids all containing a C18-sphingosine base and
various fatty acids. The oligosaccharide chain of these
glycosphingolipids consists of two N-acetylgalactosamine,
two galactose and one glucose residue. In particular the
major peaks at m/z 1,564.9 and 1,542.2 appear to correspond
to the [M ? Na]? and [M ? H]? ions of species containing
saturated fatty acids with 24 carbon atoms respectively,
while that at m/z 1,514.9 to the [M ? H]? ion of a specie
having C22:0. The minor peaks at m/z 1,487.5 and 1,592.7
can be attributed to the [M ? H]? ion of Gb5 with C20:0
and to the [M ? Na]? ion of Gb5 with C26:0, respectively.
Galactocerebrosides, giving signals in the range m/z
800–850, were not well distinct from the background.
The lipid extract obtained from the membrane fraction
enriched in cilia was also analyzed by MALDI-TOF-MS;
the mass spectra obtained (negative and positive mode)
Table 2 MALDI-TOF signals of phospholipids of pig olfactory epithelium
Class of phospholipid Ion
Total fatty acid carbon n:n of double bonds
(32:0) (32:1) (34:1) (34:2) (36:0) (36:2) (36:4) (38:4) (40:4) (16:0) (18:0) (22:0) (24:0) (36:4)
(36:4)
Ptd2Gro
[M - H]-
–
–
–
–
–
–
–
–
–
–
–
–
–
1,448
PtdOH
[M - H]-
–
–
–
–
701
–
–
723
–
–
–
–
–
–
PtdEtn
[M - H]
-
–
–
–
–
–
742
–
766
–
–
–
–
–
–
p-PtdEtn
[M - H]-
–
–
–
–
–
–
–
750
–
–
–
–
–
–
PtdCho
[M ? H]? 734
–
760
–
–
786
–
810
–
–
–
–
–
–
PtdSer
[M ? H]?
–
734
–
–
–
–
–
–
–
–
–
–
–
–
PtdSer
[M - H]-
–
–
–
760
788
–
–
810
838
–
–
–
–
–
PtdIns
[M - H]
-
–
–
–
–
–
–
857
885
–
–
–
–
–
–
CerPCho
[M - H]?
–
–
–
–
–
–
–
–
–
703
731
787
815
–
Data reported are obtained from the analysis of total lipid extracts and bands isolated from preparative TLC. The m/z values are reported. The
numbers (x:y) denote the total length and number of double bonds of both acyl chains, respectively, except for PtdEtn plasmalogen species
(denoted with p-PtdEtn), in which the acyl chain at the sn-1 position is replaced with an alkenyl. For CerPCho species, the numbers in brackets
correspond to the length and number of double bonds of the acyl chain, attached to the sphingosine base. For Ptd2Gro species, the numbers refer
to the two pairs of acyl chains
123
600
Lipids (2010) 45:593–602
Table 3 MALDI-TOF signals of glycolipids of pig olfactory epithelium
Class of
glycolipid
Ion
S-GalCer 24:0
[M - H]-
S-GalCer 26:0
[M - H]-
Gb5 26:0
Gb5 24:0
m/z
No. of
sulfated
residue
Chain length (carbon n:n of double bonds)
Oligosaccharides (no. of residues)
Sphingoid
Acyl
Glc
Gal
GalNAc
906.6
1
18:1
24:0h
–
1
–
934.5
1
18:1
26:0h
–
1
–
[M ? Na]?
[M ? Na]?
1,592.7
1,564.9
–
–
18:0
18:0
26:0
24:0
1
1
2
2
2
2
Gb5 24:0
[M ? H]?
1,542.2
–
18:0
24:0
1
2
2
Gb5 22:0
[M ? H]?
1,514.9
–
18:0
22:0
1
2
2
Gb5 20:0
[M ? H]?
1,487.5
–
18:0
20:0
1
2
2
S-GalCer, galactosylceramide sulfate; Gb5, globopentaosylceramide; Sphingoid, amino-alcohol chain; Acyl, fatty acid; h, hydroxylated fatty
acid; Glc, glucose; Gal, galactose; GalNAc N-acetylglucosamine
Table 4
31
P-chemical shifts of standard phospholipids
Lipid
ppm
PtdCho
0.115
PtdIns
0.621
CerPCho
0.911
PtdSer
0.894
PtdEtn
1.066
Ptd2Gro
1.148
Chemical shifts are referenced to 85% H3PO4 as an external standard.
The samples were prepared in the Cs/EDTA analytical reagent, as
described in ‘‘Methods’’
By comparing the two NMR spectra illustrated in Fig. 6
it can be seen that PtdCho, PtdIns, PtdSer, PtdEtn and
CerPCho species were present in both the lipid extracts of
WM (a) and CM (b); PtdSer and CerPCho peaks (at about
0.9 ppm) were very close to each other and not well
resolved in the case of spectrum in panel a, because WM
possibly contain more PtdSer and CerPCho species
than CM.
The sums of the peak areas in the two spectra were
13.87 and 15.39 in CM and WM, respectively; this indicates that the total lipid phosphorus per mg of total lipid
extract in CM was slightly lower than in WM and represents an indirect indication of the presence of an higher
proportion of glycosphingolipids in cilia, compared to
whole membranes. The last main peak at 1.05 ppm was
assigned to a plasmenyl-PtdEtn lipid, in agreement with a
previous literature report [19]; also the small, but clearly
visible peak at 0.18 ppm can be attributed to plasmalogen
PtdCho species, as well [19]. The Ptd2Gro peak in the
NMR profile of whole membranes was absent because its
relative proportion in the lipid extract was below the sensitivity limit of NMR analysis.
123
Fig. 6 31P-NMR spectra of the lipid extract of whole-tissue (a) and
enriched in olfactory cilia (b) membranes. The areas of peaks are
reported below the x-axis
The differences in the area peaks of the two NMR
spectra indicate that the proportions of various lipid classes
in the two lipid extracts were different. The area ratios
PtdEtn species/(PtdSer ? CerPCho) in CM and WM were
1.23 and 1.62 respectively, indicating that the proportion of
PtdEtn species (as the sum of diacyl- and acyl-alkyl forms)
was lower in CM extract in agreement with results from
TLC analyses (Figs. 2, 3).
Lipids (2010) 45:593–602
Discussion
Lipid research can offer important elements to complete
our understanding of the genesis of sensory perception
pathologies. For example anosmia, i.e. the inability to
perceive odors, can arise from loss of olfactory cilia or
impairment in the olfactory signaling cascade [20]. The
complete comprehension of the mechanisms of ciliogenesis
and assembly of membrane signaling molecules requires an
integration of lipid and protein studies.
The present paper describes the results of a study on
cellular lipids of porcine olfactory mucosa, previously
selected to investigate the response of olfactory cilia to
explosives, in the frame of an investigation to understand
the molecular basis of the ability of an animal nose to
detect buried landmines [21]. In the pig nose the olfactory
mucosa is well distinct from the respiratory mucosa, while
in other mammals technical difficulties are often involved
in obtaining olfactory tissue from ethmoids without concomitant excision of non-olfactory tissue [7].
As the olfactory mucosa consists of three cell types
(olfactory sensory neurons, supporting and basal cells),
results of whole membrane lipid analyses refer to the
average lipid composition of membranes arising from the
different cell types in the epithelium.
A combination of a number of different analytical
techniques was used to analyze in detail the lipids of
olfactory neuroepithelium together with the novel MALDITOF-MS approach based on the use of the versatile matrix
9-aminoacridine [15].
The results shown here indicate that the lipid composition
of olfactory mucosa in pig is similar to that of bovine and rat
[6, 7]. Phospholipids account for about 85–90% (by weight)
of the total lipid of mucosa, with zwitterionic lipids (PtdEtn,
PtdCho and CerPCho) being more abundant (about 70%)
than anionic species (PtdSer, PtdIns, Ptd2Gro, PtdOH). Most
phospholipids show a polyunsaturated fatty acid content
with the arachidonic acid (20:4) residue predominating. The
fatty acids in sphingomyelin, however, are totally saturated
and include C16:0, C18:0, C22:0, and C24:0 chains.
Although separation of acyl-acyl from alkyl-acyl forms of
phospholipids cannot be easily achieved by TLC, NMR analyses reveal that peak areas of alkyl-acyl PtdEtn almost equal
the diacyl-PtdEtn species, as previously reported for mammalian brain tissue [22]. MALDI-TOF-MS analyses indicate
that the main PtdEtn species contain C18 and C20 chains.
As regards glycosphingolipids, sulfoglycosphingolipids
having C24:0 hydroxy-fatty acid and the sphingoid
4-sphingenine (d18:1), and pentaosylceramides containing
a C18-sphingosine base and various fatty acids, were only
detected by MALDI-TOF analysis.
Here information on the lipid composition of a membrane fraction enriched in olfactory cilia is also reported. In
601
the past biochemical exploration of the olfactory cilia was
mostly based on a membrane preparation protocol established by Chen and Lancet [9]. Thus, the examination of
the cilia preparations yields many of the molecular details,
which support the current concept for olfactory signal
transduction. In the present study we isolated a membrane
fraction enriched in ciliary membranes of about the same
quality as the analogous preparation obtained by other
authors from rat tissue [23]. The lack of cardiolipin in the
lipid extract of cilia represents a good internal index of the
absence of mitochondrial and possibly other intracellular
membranes in our preparation. However, we cannot
exclude the presence of microvillar fragments of support
cells in our ciliary membrane preparation.
In summary, the present analysis of the lipid extracts of
whole-tissue membranes and enriched ciliary membranes
shows that: (1) PtdEtn species are less abundant in ciliary
membranes than in total epithelial membranes (TLC and
NMR findings), and (2) long-chain sulfoglycosylsphingolipids (S-GalCer 26:0) are enriched in ciliary
membranes compared to the crude olfactory membranes.
The roles of sulfoglycosylsphingolipids in metabolism and
functions of nervous tissue have been recently reviewed
[24]. In principle, given the importance of lipids in signaling, any difference in the lipid bilayer composition might be
considered a factor that can affect the membrane transduction properties. A decrease of membrane PtdEtn content
was previously described in brain pathologies [22]. In
olfactory cilia a decrease of PtdEtn level implies an increase
in other membrane lipids, such as CerPCho, PtdSer and
PtdIns, having a well known role in the assembly of lipid
rafts (CerPCho, PtdSer) and signal transduction (PtdIns).
On the other hand, PtdEtn is preferentially located in the
inner membrane leaflet, where it is associated with acidic
phospholipids such as PtdIns [25]. Being cone-shaped
phospholipid, PtdEtn can influence the determination of
membrane curvature; it has been reported that intracellular
tubular membranes of Golgi apparatus have a minor
amount of PtdEtn compared to plasma membrane [25]. The
possibility of a correlation between the low PtdEtn content
and the high curvature in cross section of tubules of
olfactory cilia remains to be investigated.
Although the presence of cholesterol-rich rafts in the
olfactory cilia has been indirectly suggested by some biochemical studies [4, 5], here no cholesterol enrichment was
found in the lipid extract of the ciliary membranes. However it should be considered that the analysis of the CM
lipid extract gives information on the average lipid composition of the membrane lining the long (up to 100 lm)
tubular structure of cilia, which presumably consist of
different biochemical and functional domains.
In particular as it has been shown that cholesterol is
absent at the necklace (or base) of the ciliary structure [26,
123
602
27], cholesterol in the CM lipid extract might represent the
average of cholesterol in the proximal and distal tubular
membranes. In conclusion, we cannot exclude the presence
of cholesterol-rich domains (i.e., rafts) in ciliary membranes on the basis of the lack of cholesterol enrichment in
the lipid extract of ciliary membranes.
Another element of complexity in the interpretation of
lipid data arises from the recent finding that the chemosensory apparatus of cilia is also present at the level of the
emergency cone of the sensory olfactory axon [28].
Here besides reporting the first lipidomic data on
olfactory mucosa in toto, we show for the first time the
presence of different lipid domains in the membranes of
neuroepithelial olfactory cells. Our study complements
recent proteomic studies, in which many ciliary proteins,
that mediate chemo-electrical transduction, amplification
and adaptation of the primary sensory signal, have been
identified [29–32].
Acknowledgments We thank F. Naso of the University of Bari for
the use of the MALDI-TOF instrument. This work was supported by
the Italian Ministry of Defence (Contract n. 685/18.12.2003), by
Regione Puglia (Grant code 15, Sens&MicroLab) and by Fondazione
Cassa di Risparmio di Puglia, Bari, Italy.
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