Research
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Acta Biochim Biophys
Sin 2005,37:515-524
doi:10.1111/j.1745-7270.2005.00073.x
A Lipidomic Study of the Effects of N-methyl-N’-nitro-N-nitrosoguanidine
on Sphingomyelin Metabolism
Yun HUANG&, Jing SHEN#,
Ting WANG, Yan-Ke YU, Fanqing F. CHEN1, and Jun YANG*
Department of
Pathology and Pathophysiology, Center for Environmental Genomics, Zhejiang
University School of Medicine, Hangzhou 310031, China;
1 Molecular Biology Branch,
Life Science Division, Lawrence Berkeley National Laboratory, University of
California at Berkeley, Berkeley, CA 94720, USA
Received: March 20,
2005
Accepted: May 23,
2005
& Present address: Department
of Chemistry, Georgia State University, Atlanta, GA 30303, USA
# These authors
contributed equally to this work
*Corresponding
author: Tel/Fax, 86-571-87217149; E-mail, [email protected]
Abstract Systems biology is a new
and rapidly developing research area in which, by quantitatively describing the
interaction among all the individual components of a cell, a systems-level
understanding of a biological response can be achieved. Therefore, it requires
high-throughput measurement technologies for biological molecules, such as
genomic and proteomic approaches for DNA/RNA and protein, respectively.
Recently, a new concept, lipidomics, which utilizes the mass spectrometry (MS)
method for lipid analysis, has been proposed. Using this lipidomic approach,
the effects of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)
on sphingomyelin metabolism, a major class of sphingolipids, were evaluated.
Sphingomyelin molecules were extracted from cells and analyzed by
matrix-assisted laser desorption ionization-time of flight MS. It was found
that MNNG induced profound changes in sphingomyelin metabolism, including the
appearance of some new sphingomyelin species and the disappearance of some
others, and the concentrations of several sphingomyelin species also changed.
This was accompanied by the redistribution of acid sphingomyelinase (ASM), a
key player in sphingomyelin metabolism. On the other hand, imipramine, an
inhibitor of ASM, caused the accumulation of sphingomyelin. It also prevented
some of the effects of MNNG, as well as the redistribution of ASM. Taken
together, these data suggested that the lipidomic approach is highly effective
for the systematic analysis of cellular lipids metabolism.
Key words lipidomics; mass
spectrometry; ceramide; sphingomyelin; acid sphingomyelinase
The completion of the human genome project has led to a revolution
in the world of biological science: the generation of “genomics”.
Following this event, “omics” in other disciplines also emerged, such
as proteomics, metabonomics, toxicogenomics and pharmacogenomics [1,2]. All of
these “omics”, genomics and proteomics in particular, form the
foundation for a new research field, systems biology. The goal of systems
biology is to formulate a computational/mathematical model that describes the
structure of the system and its response to individual perturbations through
the monitoring of systematic changes of all cellular components (genes,
proteins, or signaling pathways) in response to any type of perturbation
(biological, genetic, or chemical) [3,4]. Therefore, it requires certain
technical approaches which can define many cellular molecules at multiple
levels; microarray for DNA analysis in genomics and 2-dimensional (2-D) gel
electrophoresis combined with mass spectrometry (MS) for protein analysis in
proteomics are just such methods. It has been gradually recognized that studying DNA and protein alone
does not engender a full understanding of a complex biological response, as
other major cellular constituents including lipids and carbohydrates are also
involved in many physiological processes. Consequently, the lack of such
information would hamper the construction of a computational model for systems
biology. Recently, a new concept, “lipidomics”, has been proposed
[5,6]. Lipidomics is a comprehensive analysis of lipid molecules which, in
combination with genomics and proteomics, is essential for the understanding of
cellular physiology and pathology. Consequently, lipid biology has become a
major research target of the postgenomic revolution and systems biology [7].Lipids are crucial structural/functional components of cells. As
structural material, they not only provide a physical barrier for cells, but
also provide a platform (or lipid raft) for membrane protein-protein
interaction. Even more importantly, many lipid species have distinct cellular
functions. For example, diacylglycerol, ceramides, eicosanoids and lysolipids
are all second messengers which participate in various cellular events such as
growth, proliferation, differentiation and cell death [8]. Sphingolipids are a
group of sphingoid-based lipids which are gaining increasing attention from
researchers. They are the major components for lipid raft. Furthermore,
sphingolipids and their metabolites are involved in many important signal
transduction pathways which regulate such cellular processes as cell cycle
arrest or apoptosis, proliferation and calcium homeostasis, as well as cancer
development, multidrug resistance, and viral or bacterial infection processes
[9]. Clearly, the importance of this group of lipids should not be
underestimated.Unfortunately, the study of lipids is far behind those of genes and
proteins. One major obstacle is the lack of high-throughput technologies in
lipid analysis. The traditional methods, such as isotope labeling, thin-layer
chromatography and high
performance liquid chromatography, could provide some
useful information, but are far from adequate. Nevertheless, until the
application of MS in sphingolipid study does a great amount of information is
generated. Compared with traditional methods, MS analysis is more accurate,
less labor-intensive and, most of all, can identify the molecular species of
each class of lipids [10,11]. In our previous studies, using isotope labeling
methods as well as matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) MS, it has been shown that N-methyl-N’-nitro-N-nitrosoguanidine
(MNNG), an alkylating agent which is a potent carcinogen, can affect ceramide
metabolism [12,13]. Sphingomyelin is another important sphingolipid species
closely related to ceramide metabolism, for example, sphingomyelin can be
hydrolyzed to generate ceramide [9]. Therefore, it is quite reasonable to
speculate that MNNG would also affect sphingomyelin metabolism. In this research, we investigated the effects of MNNG on
sphingomyelin metabolism. In addition, the cellular distribution of acid
sphingomyelinase (ASM), a key enzyme in sphingomyelin metabolism, was also determined.
As reported here, MNNG induced the generation/loss of some sphingomyelin
species, as well as the increase/decrease of other sphingomyelin species.
Materials and Methods
Cell culture and reagents
Human amnion FL cells were cultured in Eagle’s minimum essential
medium (EMEM; Invitrogen, Carlsbad, USA) containing 10% fetal bovine serum,
supplemented with 100 U/ml penicillin, 100 U/ml streptomycin and 0.03% L-glutamine
in a humidified incubator at 37 ?C with 5% CO2. MNNG (Sigma,
St. Louis, USA) was dissolved in dimethylsulfoxide (DMSO) as a 10 mM stock.
Imipramine (Sigma) was also dissolved in DMSO as a 50 mM stock. For MNNG
treatment, cells were treated with 10 mM of MNNG for 20 min. DMSO-treated or untreated
cells were used as solvent control or blank control, respectively.D-sphingosine, N-acetyl-D-sphingosine
(C2-ceramide), N-hexanoyl-D-sphingosine (C6-ceramide), N-octanoyl-D-sphingosine
(C8-ceramide), D–threo-ceramide C8, dihydrosphingosine, C2-dihydroceramide,
C6-dihydroceramide, and C8-dihydroceramide were all purchased from Sigma; and
each was dissolved following the manufacturer’s instructions.
Immunofluorescent microscopy
The translocation of ASM was observed by immunofluorescent microscopy
as described before [14]. Briefly, 1?105 FL
cells were seeded into a 6-well culture plate with a glass cover slip in each
well. After MNNG (10 mM) treatment for 20 min, cells were fixed and permeated with 100%
ice-cold methanol for 5 min, followed by blocking in a blocking solution
(Zymed Laboratories Inc., San Francisco, USA) for 2 h. The plate was washed
with PBS three times and the polyclonal rabbit anti-ASM antibody (1:200; Santa
Cruz Biotechnology, Santa Cruz, USA) was added and incubated for 90 min.
Cy3-labeled goat anti-rabbit secondary antibody (1:200; Boster Biological
Technology Limited, Wuhan, China) was then added to the plate and incubated for
1 h. These cells were then washed and stained with 500 ng/ml FITC-cholera toxin
B (Sigma) for 1 h. The cover slip was removed from the plate, mounted onto a
glass slide, observed with an Olympus AX70 fluorescent microscope (Olympus,
Tokyo, Japan), and analyzed using Image-Pro Plus software (MediaCybernetics,
Silver Spring, USA). For imipramine treatment, cells were pre-incubated with 50
mM
imipramine for 1 h before adding MNNG.
Sphingomyelin extraction and MALDI-TOF MS
Sphingomyelin was extracted as described before [12]. In short,
approximately 4?107
cells were resolved in 500 ml chloroform:methanol (2:1, V/V). 1 ml H2O
was then added to each sample. The mixed samples were centrifuged at 4770 g
for 15 min and the lower phase was dried by vacuum centrifugation in a
centrifugal evaporator (Speed-Vac, Thermo Savant, Holbrook, USA). Then, 500 ml methanol
containing 0.1 M NaOH was added into each tube at 55 ?C for 1 h to decompose
glycerophospholipids. After neutralization with 100 ml methanol containing 1 M
HCl, 500 ml hexane and one drop of water were added to each sample. The mixture
was then centrifuged again at 4770 g for 15 min and the lower phase was
dried in a centrifugal evaporator after the upper phase was removed. The
residue was mixed with 0.8 ml theoretical lower phase
(chloroform:methanol:water, 86:14:1, V/V) and 0.2 ml theoretical
upper phase (chloroform:methanol:water, 3:48:47, V/V) for the
Folch partition, and centrifuged at 4770 g for 15 min. The lower phase
was evaporated in a centrifugal evaporator after removing the upper phase to
discard the salt. The residue crude sphingomyelin was stored at –70 ?C.For MALDI-TOF MS analysis, each sample was dissolved in 5 ml
chloroform:methanol (2:1, V/V), followed by the addition of 5 ml matrix
solution, ethylacetate containing 0.5 M 2,5-dihydroxyl-benzoic acid (2,5-DHB;
Sigma) and 0.1% TFA, in a 0.5 ml Eppendorf tube. The tube was agitated
vigorously on a vortex mixer then centrifuged in a microcentrifuge for 1 min.
Then, 1 ml of mixture was directly added to the sample plate and rapidly
dried under a warm stream of air in order to remove the organic solvent within
seconds. All samples were analyzed using a Voyager-DE STR MALDI-TOF mass
spectrometer (ABI Applied Biosystem, Framingham, USA) with a 337 nm N2 UV
laser. The mass spectra of the samples were obtained in positive ion mode.
Mass/charge (m/z) ratios were measured in the reflector/delayed
extraction mode with an accelerating voltage of 20 kV, grid voltage of 67% and
delay time of 100 ns. C2-dihydroceramide (MW 343.6) was used to calibrate the
instrument. All sample lipid spectra were acquired using a low-mass gate at 400
Da. For each sample, 6 or 7 spectra were obtained; only when a peak appeared in
at least 5 spectra with relatively stable intensity was it considered a
candidate for analysis. All MS data were analyzed as described before [15,16].
Results
Establishment of MS data analysis protocol
In order to establish a working protocol for analyzing MS data for
sphingolipids, up to 10 different sphingolipid species (natural or synthetic) were
subjected to MS, and the major peaks from resulting mass spectra were
calculated to deduce the possible chemical structures. The major peaks from
two sphingolipids molecules, C8-ceramide and C8-dihydroceramide, are listed in Table
1 and Table 2, respectively. 25 major peaks were generated by
C8-ceramide during the ionization process, of which most were from the matrix
2,5-DHB. m/z 425 (425.6896) corresponded to the intact
C8-ceramide. However, the relative intensity of this peak was only 14.17%; on
the other hand, m/z 407 (407.7233) had a relative intensity of
100%. Based on calculation it was concluded that m/z 407 could
stand for the fragment of C8-ceramide with an H2O loss,
suggesting that most C8-ceramide lost one molecule of water
during ionization. Further chemical structural analysis gave two possible
structures for this fragment [Fig. 1(A)]. The ionization process can
even break the whole octal carbon sidechain away from a small portion (4.12%)
of C8-ceramide, resulting in the formation of a D-sphingosine-like
fragment, which corresponded to m/z 281 (281.3613). Two isotope
peaks were also present for m/z 407 and one for 425 (Table 1).
Similar analysis was also conducted for C8-dihydroceramide (Table 2),
and the possible chemical structures for some fragments are depicted in Fig.
1(B). Together, these processes formulated the basic protocol for
sphingolipids MS data analysis.
MNNG induces dramatic changes in sphingomyelin metabolism
Previously we have shown that MNNG can induce changes in ceramide
metabolism [12,13]. As sphingomyelin is closely associated with ceramide, we
further examined the cellular sphingomyelin metabolism using MALDI-TOF MS. The
major sphingomyelin peaks obtained from control, DMSO-treated, and MNNG-treated
cells are listed in Table 3. It was found that while DMSO had only a
minor effect on sphingomyelin metabolism, there were significant differences
between MNNG-treated and control samples for sphingomyelin. For example, m/z
778 was not present in control but appeared after MNNG treatment; whereas m/z
782 showed up in control but disappeared after MNNG treatment (Table 3).
In addition, the concentrations of several sphingomyelin species, including m/z
770, 784, 805 and 814, were increased. The mass spectra data for some
sphingomyelin species [Fig. 2(A), m/z 782, 784, 805 and
814] and possible structures for some of the identified sphingomyelin species
are also presented (Table 4).
MNNG induces the redistribution of ASM
ASM is responsible for hydrolyzing sphingomyelin to generate
ceramide, and its translocation is usually associated with its activation [17–19]. Using immunofluorescent microscopy, the distribution of ASM and
its relationship with lipid rafts were studied. ASM exhibited a diffused, even
distribution in control cells [Fig. 3(A)] and DMSO solvent control (data
not shown). However, MNNG treatment caused the “polarization” of ASM,
which concentrated on one side of the cell [Fig. 3(A)]. In addition, ASM
colocalized with lipid raft, which was labeled by cholera toxin B. This
observation implied that ASM might be involved in the altered sphingomyelin
metabolism.
Imipramine induces the accumulation of sphingomyelin and inhibits
some of the effects of MNNG on sphingomyelin
Imipramine is known to inhibit ASM activity [20]. MNNG-induced
changes in sphingomyelin may be a result of ASM activation, therefore cells were
pre-incubated with imipramine followed with MNNG treatment and, after
sphingomyelin extraction, the mass spectra were compared with those without
imipramine. It was found that imipramine alone could cause the accumulation of
several sphingomyelin species, indicating that it inhibited the hydrolysis of
sphingomyelin (Table 3). Furthermore, it diminished some effects of MNNG
on sphingomyelin. For example, the increases of m/z 770 and 784
by MNNG treatment were reversed by imipramine pre-incubation, and the
disappeared m/z 782 was restored [Table 3, Fig. 2(B)].
Furthermore, imipramine also prevented the polarization of ASM induced by MNNG,
implying the inactivation of ASM [Fig. 3(B)].
Discussion
Many sphingolipid molecules, such as ceramide, sphingosine and
sphingosine-1-phosphate, are increasingly recognized as important modulators
of many cellular processes. For example, ceramide has been shown to function
as a second messenger for Fas, tumor necrosis factor, interleukin (IL-1) and
other cytokines, as well as many other extracellular stimuli, usually with the
result of either cell cycle arrest or apoptosis [9,13,21,22]. However, unlike
nucleotides and proteins, lipids and sphingolipids, have long been a group of
molecules that are difficult to study. Sphingolipids were named after the
famous Egyptian statue “Sphinx” for their mystical properties. The
bottleneck in research was due to the lack of suitable technologies for
analyzing the vast number of lipid species, even less the high-throughput
technology for systems biology. The breakthrough came after the application of MS to lipid study, in
which many molecules from the lipidome could be directly characterized and
quantitated [8,10,11]. Using this method, we have shown that MNNG can affect
the metabolism of a major sphingolipid species, ceramide [12]. This change of
metabolism is associated with some of the cellular effects of MNNG, such as
membrane receptor clustering [12]. In this study, we further evaluated the
metabolism of sphingomyelin, which can be hydrolyzed by ASM to generate
ceramide [9]. It was found that, similar to ceramide, sphingomyelin metabolism
was also affected by MNNG treatment (Table 3), indicating that MNNG may
have a global effect on sphingolipids metabolism.More importantly, using this MS approach, the different
sphingomyelin species could be identified, and the changes for each species
measured. For instance, eight distinct m/z ratios were identified, with
each m/z ratio representing one or more possible molecular
structures (Table 3). The differences in sidechain length, as well as
the number of unsaturated bonds, may influence the function of a specific
lipid molecule. Therefore, this type of analysis provides invaluable
information that cannot be obtained using traditional methods. The applicability of this technique was further validated by the
imipramine experiment. As an inhibitor for ASM, it was expected that imipramine
would inhibit the hydrolysis of sphingomyelin, thus increasing the cellular
content of sphingomyelin. Indeed it was found that imipramine treatment
caused the accumulation of several sphingomyelin species, particularly m/z
748 and 782 [Table 3, Fig. 2(B)]. In addition, imipramine
pre-incubation interfered with the effect of MNNG on sphingomyelin, indicating
that MNNG probably elicited its effect through the action of ASM. This was also
supported by the immunfluorescent microscopy data, as MNNG treatment triggered
the relocation of ASM, while imipramine prevented it (Fig. 3). Some problems do exist for the MS method. For example, except for a
few m/z ratios, exact chemical structures could not be deduced
precisely; instead, several possibilities were formulated. Furthermore, when
standard sphingolipids were subjected to MALDI-TOF analysis, several fragments
were generated for each standard (for example, m/z 407 and 281
for C8-ceramide). It would be difficult to tell if these fragments were the
original forms presented in the sample or just fragments generated from other
molecules during the ionization process. The presence of matrix peaks
complicates the analysis even further. Finally, the reproducibility of mass
spectra should be carefully handled. Mass spectrometry is a very sensitive
method and efforts should be taken to minimize the variations which might
affect the analyses. Compared with MALDI-TOF, liquid chromatography-electrospray
ionization MS (LC-ESI MS) may prove to be a better solution. First, there is no
need for matrices in the analysis. Secondly, its “soft” ionization
process, generally, would not fragment the samples. Therefore, LC-ESI MS has
far less “noise” than MALDI-TOF MS. Nevertheless, there is the
possibility that two molecules have distinct structures but the same molecular
weight. To solve this problem, Han and Cheng developed a 2-D ESI MS/MS method
[10]. Through lipid class-selective intrasource ionization and subsequent
analysis of 2-D cross-peak intensities, the chemical identity and mass
composition of individual molecular species of most lipid classes can be
determined [10]. In summary, the data presented here demonstrated that MALDI-TOF MS
is a powerful tool in lipid research. Together with the 2-D ESI MS/MS method,
these techniques provide a strong foundation for the automated analysis of
lipid mass spectra data, which will help to push the study of systems biology
to a new level.
Acknowledgements
The authors gratefully thank Dr. T. Taketomi for providing detailed instructions for analyzing
the MALDI-TOF mass spectrometry data, and Dr. X. Han for the helpful discussion regarding lipidomics.
References
1 Pognan F. Genomics, proteomics and metabonomics in
toxicology: Hopefully not ‘fashionomics’. Pharmacogenomics 2004, 5: 879–893
2 Kramer R, Cohen D. Functional genomics to new drug
targets. Nat Rev Drug Discov 2004, 3: 965–972
3 Aggarwal K, Lee KH. Functional genomics and proteomics
as a foundation for systems biology. Brief Funct Genomic Proteomic 2003, 2: 175–184
4 Kell DB. Metabolomics and systems biology: Making sense
of the soup. Curr Opin Microbiol 2004, 7: 296–307
5 Han X, Gross RW. Global analyses of cellular lipidomes
directly from crude extracts of biological samples by ESI mass spectrometry: A
bridge to lipidomics. J Lipid Res 2003, 44: 1071–1079
6 Lagarde M, Geloen A, Record M, Vance D, Spener F.
Lipidomics is emerging. Biochim Biophys Acta 2003, 1634: 61
7 Fahy E, Subramaniam S, Brown HA, Glass CK, Merrill AH
Jr, Murphy RC, Raetz CR et al. A comprehensive classification system for
lipids. J Lipid Res 2005, 46: 839–862
8 Han X, Yang J, Cheng H, Ye H, Gross RW. Toward
fingerprinting cellular lipidomes directly from biological samples by
two-dimensional electrospray ionization mass spectrometry. Anal Biochem 2004,
330: 317–331
9 Yang J, Yu Y, Sun S, Duerksen-Hughes PJ. Ceramide and
other sphingolipids in cellular responses. Cell Biochem Biophys 2004, 40: 323–350
10 Han X, Cheng H. Characterization and direct quantitation of
cerebroside molecular species from lipid extracts by shotgun lipidomics. J
Lipid Res 2005, 46: 163–175
11 Han X, Gross RW. Shotgun lipidomics: Electrospray ionization
mass spectrometric analysis and quantitation of cellular lipidomes directly
from crude extracts of biological samples. Mass Spectrom Rev 2005, 24: 367–412
12 Huang Y, Yang J, Shen J, Chen F, Yu Y. Sphingolipids are
involved in N-methyl-N‘-nitro-N-nitrosoguanidine-induced epidermal
growth factor receptor clustering. Biochem Biophys Res Commun 2005, 330: 430–438
13 Yang J, Duerksen-Hughes PJ. Activation of a p53-independent,
sphingolipid-mediated cytolytic pathway in p53-negative mouse fibroblast cells
treated with N-methyl-N-nitro-N-nitrosoguanidine. J Biol Chem 2001, 276: 27129–27135
14 Gao Z, Yang J, Huang Y, Yu Y. N-methyl-N‘-nitro-N-nitrosoguanidine
interferes with the epidermal growth factor receptor-mediated signaling
pathway. Mutat Res 2005, 570: 175–184
15 Fujiwaki T, Yamaguchi S, Tasaka M, Sakura N, Taketomi T.
Application of delayed extraction-matrix-assisted laser desorption ionization
time-of-flight mass spectrometry for analysis of sphingolipids in pericardial
fluid, peritoneal fluid and serum from Gaucher disease patients. J Chromatogr B
Analyt Technol Biomed Life Sci 2002, 776: 115–123
16 Fujiwaki T, Yamaguchi S, Sukegawa K, Taketomi T. Application
of delayed extraction matrix-assisted laser desorption ionization
time-of-flight mass spectrometry for analysis of sphingolipids in cultured
skin fibroblasts from sphingolipidosis patients. Brain Dev 2002, 24: 170–173
17 Grassme H, Cremesti A, Kolesnick R, Gulbins E.
Ceramide-mediated clustering is required for CD95-DISC formation. Oncogene
2003, 22: 5457–5470
18 Gulbins E, Grassme H. Ceramide and cell death receptor
clustering. Biochim Biophys Acta 2002, 1585: 139–145
19 Gulbins E. Regulation of death receptor signaling and
apoptosis by ceramide. Pharmacol Res 2003, 47: 393–399
20 Lacour S, Hammann A, Grazide S, Lagadic-Gossmann D, Athias A,
Sergent O, Laurent G et al. Cisplatin-induced CD95 redistribution into
membrane lipid rafts of HT29 human colon cancer cells. Cancer Res 2004, 64:
3593–3598
21 Pru JK, Hendry IR, Davis JS, Rueda BR. Soluble Fas ligand
activates the sphingomyelin pathway and induces apoptosis in luteal
steroidogenic cells independently of stress-activated p38(MAPK). Endocrinology
2002, 143: 4350–4357
22 Luberto C, Hassler DF, Signorelli P, Okamoto Y, Sawai H, Boros
E, Hazen-Martin DJ et al. Inhibition of tumor necrosis factor-induced
cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase. J Biol
Chem 2002, 277: 41128–41139