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Acta Biochim Biophys
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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 ºC 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 ºC 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 ºC.
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.
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