Glycobiology Advance Access originally published online on December 3, 2007
Glycobiology 2008 18(2):158-165; doi:10.1093/glycob/cwm129
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Communication |
Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry
2 Department of Melanoma Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77054, USA
3 Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, Göteborg University, SE-40530 Göteborg, Sweden
4 Howard Hughes Medical Institute, Department of Pathology and Committee of Immunology, University of Chicago, Chicago, IL 60637, USA
5 Department of Chemistry, University of New Hampshire, Durham, NH 03824-3598, USA
1 To whom correspondence should be addressed: Tel: +1-713-792-3134; Fax: +1-713-563-3424; e-mail: dzhou{at}mdanderson.org and Tel: +1-603-862-2529; Fax: +1-603-862-4278; e-mail: slevery{at}cisunix.unh.edu
Received on September 30, 2007; revised on November 25, 2007; accepted on November 28, 2007
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Abstract |
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 Top Abstract IntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Glycosphingolipids serve as ligands for receptors involved in signal transduction and immune recognition, as exemplified by isoglobotrihexosylceramide, an antigenic ligand for T cell receptors. Mechanistic studies on the regulation of isoglobotrihexosylceramide require biochemical measurement of its lysosomal precursor, isoglobotetraglycosylceramide. It remains a challenge to distinguish between complex tetraglycosylceramide glycosphingolipid isomers with the same sugar components but diverse internal linkages. Here we established a simple and sensitive method to separate globo- and isoglobotetraglycosylceramide by MS5 ion trap mass spectrometry, and report the identification of isoglobotetraglycosylceramide in a CHO cell line transfected by iGb3 synthase, as well as in human thymus.
Key words: CD1d / glycosphingolipid / ion trap MS / isoglobotetraglycosylceramide / natural killer T cells
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Introduction |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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The role of glycoconjugates in cell signaling has emerged from the combined effort of biologists, analytical chemists and synthetic chemists (Zhou 2006a
, 2006b). The concept that glycans are ligands for biological receptors underlies current functional glycomics research initiatives. In contrast to protein or nucleotide ligands, the creation of glycan ligands depends on an "assembly line" (Kornfeld R and Kornfeld S 1985), i.e., it requires multiple enzymes for stepwise assembly. The regulation of glycan ligands is dependent not only on the transcriptional regulation of multiple glyco-enzymes, but also on the interaction of these glyco-enzymes with chaperone or activator proteins in different intracellular compartments, and the availability of substrates for glyco-enzymes. Thus, the regulation of expression of a specific glycan ligand is much more complex than that of proteins and nucleotides.
Such glycans serving as biological ligands include the glycosphingolipid (GSL) recognized by natural killer T (NKT) cells. NKT cells are a subset of innate-like T cells that carry the surface markers of both T cells and NK cells (Kronenberg and Gapin 2002; Taniguchi et al. 2003; Pear et al. 2004; Bendelac et al. 2007). NKT cells express very restricted, evolutionarily conserved, and germ-line-encoded T cell receptors (TCRs), as compared to the extremely diverse, recombined TCRs expressed by conventional T cells. In contrast to conventional T cells which recognize peptide antigens presented by MHC molecules, NKT cells recognize glycolipid antigens presented by a non-MHC-encoded, nonpolymorphic, MHC-like antigen-presenting molecule, CD1d. The activation of NKT cells is strictly dependent on TCR stimulation, but is inordinately rapid, leading to cytokine secretion within 2 h. Thus, like NK cells, NKT cells play critical "jump starting" functions in antimicrobial and antitumor immunity, through mechanisms that are only partially understood. NKT cells activate the professional antigen-presenting cells (dendritic cells) to enhance their function of antigen presentation. The molecular interaction between NKT cells and dendritic cells remains partially understood. It has been demonstrated that TCR recognition of glycolipid antigens presented by CD1d in dendritic cells is essential for initiating the crosstalk between NKT cells and dendritic cells. For example, in a study on tumor progression in mice, NKT cells interacted with dendritic cells in the tumor microenvironment, and the activation of NKT cells by tumor-derived ligands promoted the activation of adaptive immune response (CD4 and CD8 responses), which caused rejection of tumor (Smyth et al. 2000). In another study, NKT cells were activated by dendritic cell-derived glycolipid antigens, and protected the mice from bacteria infection (Mattner et al. 2005).
The identities of endogenous NKT ligands have been an enigma for more than a decade. Genetic evidence suggested that these ligands are GSLs derived from glucosylceramide (Stanic et al. 2003). Zhou et al. first identified a neutral GSL, isoglobo- triosylceramde (iGb3), as a natural ligand (Zhou et al. 2004; Zhou 2006b). This finding was confirmed by multiple independent studies showing that iGb3 stimulated a variety of mouse and human NKT clones representing a majority of invariant NKT population (Schumann et al. 2006; Xia et al. 2006; Scott-Browne et al. 2007; Yu et al. 2007). These include human NKT cell lines, mouse hybridomas generated in different laboratories and, more recently TCR-transfected lines (Behar et al. 1999; Gui et al. 2001; Scott-Browne et al. 2007). These results encouraged us to study the biochemistry of isoglobo series of GSLs in immune organs and immune cells. In view of the low abundance of T cell antigens in antigen-presenting cells, we have explored the application of ion trap mass spectrometry (MSn) technology to the analysis of complex mixtures of cellular GSLs. The principle of this method is that signature ion fragments can be generated for each specific GSL after multiple rounds of fragmentation, which allows the identification of NKT antigens such as iGb3 by mass spectrometry without purifying individual glycolipids, even in the presence of large quantity of regioisomers (Ashline et al. 2005; Lapadula et al. 2005; Zhang et al. 2005; Li et al. 2007). Here, we extended this approach to study the regioisomers iGb4 and Gb4, since iGb3 is generated in the lysosome through cleavage of iGb4 by β-hexosamindases. This new method should enhance the study of the assembly line leading to the expression of iGb3 in antigen-presenting cells. As a first step for future studies on the regulation of these biologically important GSLs, we report here the presence of iGb4 in human thymus.
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Results |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Discrimination of iGb4 from Gb4 by permethylation and multistep ion trap MSn
Previous studies showed that the terminal Gal
3Gal-1-ene disaccharide from the sodium adduct of permethylated isoglobotrihexosylceramide (iGb3, Gal3Galß4Glcβ1Cer) produces an ion trap MS4 spectrum readily distinguishable from that of the terminal Gal4Gal-1-ene disaccharide of similarly treated and analyzed globotrihexosylceramide (Gb3, Gal4Galß4Glcβ1Cer) (Ashline et al. 2005; Lapadula et al. 2005; Zhang et al. 2005; and Li et al. 2007). Since these trihexosylceramides are often observed as internal sequences in further glycosylated products, such as Gb4 and iGb4, an essential question is whether the internal Gal3/4Gal disaccharides could be isolated and discriminated by an analogous method. Compared with the nonreducing terminal disaccharides of Gb3/iGb3, one additional fragmentation step is required to generate the required internal HO-3Gal3/4Gal-1-ene precursor m/z 431. When this was fragmented in MS5, product ions were indeed observed which appeared to be diagnostic for iGb4 versus Gb4 (Scheme 1).
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Shown in Figure 1 are examples of MS5 spectra (m/z 1572
912690431) obtained from isobaric standards of Gb4 and iGb4 (Panels A and B, respectively; for permethylated sodium adducts, expected nominal, monisotopic m/z 1571; calculated monoisotopic m/z 1572.11). Product ions of m/z 431 from pure Gb4 are reproducibly obtained at m/z 209, 227, 243, 245, 301, 315, 399, and 401. Among these, two highly abundant ions are m/z 315 (base peak) and 399. One ion of intermediate abundance is at m/z 245. The remaining significant ions are at 5–20% relative abundance. The isobaric disaccharide-1-ene fragment from pure iGb4 yields highly abundant products at m/z 227, 245, 357, and 399, ions of intermediate abundance at m/z 209 and 369, and ions of the 5–15% relative abundance range at m/z 199, 225, 243, 329, and 401. These are consistent with specific glycosidic and cross-ring cleavages, along with typical neutral losses, as shown in Scheme 1. It is notable that several ions in the m/z 431 product spectrum from iGb4, including the highly abundant fragment m/z 357, are virtually absent in the corresponding spectrum from Gb4. Conversely, the most abundant ion in the m/z 431 product spectrum from Gb4, m/z 315, is barely observable above noise in the corresponding spectrum from iGb4. These can be proposed, therefore, to form the basis for discriminating the internal Gal3/4Gal linkage in binary mixtures containing isobaric Gb4/iGb4 molecular species.
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Detection of iGb4/Gb4 tetraglycosylceramides in CHO cells transfected by iGb3 synthase enzyme
To test the applicability of our method to detect iGb4 in biological samples, we analyzed a CHO cell line transfected by rat iGb3 synthase enzyme (Teneberg et al., in preparation), designated as CHO-iGb3 cells here. The CHO cell line was transfected by a mock pcDNA3.1-Hygro+ plasmid was used as a negative control, designated as CHO cells here. Neutral GSL mixtures from CHO-iGb3 and CHO cells were permethylated and analyzed by electrospray ionization linear ion trap MS (ESI-LIT-MS) as described above.
Matching sections (m/z 1540 to 1610) of ESI-LIT-MS1 profile spectra of permethylated neutral GSL fractions isolated from CHO-iGb3 and CHO cells are shown in Figure 2 (Panel A-a, B-a, respectively). In this m/z range, one would expect to observe Na+ adducts of permethylated iGb4/Gb4 tetraglycosylceramides if they are present. Among all ions in this range, a number of potential iGb4/Gb4 molecular adduct ions were observed and their sequences subsequently verified by MSn analysis. The MS1 ions at m/z 1544, 1558, 1572, 1586, 1588, 1600, and 1602 yielded the expected MS2 products consistent with iGb4/Gb4, including m/z 912 for the GalNAcβ(1–3)Gal(1–3/4)Galβ(1–4)Glc-OH glycan ion of iGb4/Gb4. The presence of iGb4/Gb4 was further confirmed by MS3 (m/z X912), MS4 (m/z X912690), and MS5 (m/z X912690431) analysis. The group of Na+-adducted tetraglycosylceramide ions at m/z 1544, 1558, 1572, 1586, and 1600 are consistent with Gb4/iGb4 having d18:1 sphingoid with 22:0, 23:0, 24:0, 25:0, and 26:0 fatty-N-acylation. The Na+-adducted tetraglycosylceramide ions at m/z 1588 and 1602 are consistent with Gb4/iGb4 having d18:0 sphingoid with 25:0, and 26:0 fatty-N-acylation. It is noteworthy that after CHO cells were transfected by iGb3 synthase, we noticed a significant increase of MS1 abundance for all tetraglycosylceramide ions.
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Most significantly, MS5 analysis showed striking differences for each individual iGb4/Gb4 isobar when comparing CHO and CHO-iGb3 cells. In CHO cells, we observed the relative abundance of fragment m/z 315 (Gb4 specific) is 100% in all the seven molecular ion species (Figure 2, MS5 spectrum of molecular ion 1572 is shown in Panel A-b). In CHO-iGb3, we found 90–100% abundance of MS5 ion m/z 357 (iGb4 specific) while a much reduced abundance of MS5 ion m/z 315 (Gb4 specific, Figure 2, Panel B-b). Besides the MS5 ion m/z 357 (iGb4 specific), the signature fragments of iGb4 at m/z 329 and 369 were also observed significantly in the MS5 spectra from CHO-iGb3 cells (Figure 2, Panel B-b). Clearly, only the CHO-iGb3 cells yielded all of the MS5 ions characteristic for iGb4, along with small amount of MS5 ions representing Gb4 pre-existing in CHO cells (Figure 3, Panel B-b). CHO cells yielded only MS5 ions representing pure Gb4 (Figure 2, Panel A-b).
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The 90–100% abundance of MS5 signature ions for iGb3, in combination with a significant increase of total iGb4/Gb4 molecular ions in CHO-iGb3 as compared with CHO in the MS1 spectrum, suggest that the majority of the tetraglycosylceramide fraction in CHO-iGb3 cells is iGb4.
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Detection of iGb4 and Gb4 in a mixture of tetraglycosylceramides isolated from human thymus |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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We next used our method to measure iGb4 in tetraglycosylceramides purified from pooled thymi from human pediatric patients. In this case, we first used an instrumental "precursor ion mapping" routine to identify all molecular adduct ions, observable in MS1, that generate m/z 912 in MS2 (Figure 3, left panel). By this method we are confident that we are not missing any MS1 ion that may represent a mixture of iGb4/Gb4. Combining precursor ion mapping with multistep ion trap MSn analysis, a series of isobaric mixtures of Gb4 and iGb4 were detected in the tetraglycosylceramide fraction isolated from human thymus. We found that the MS5 product ion m/z 357 was reliably observed as a signature of iGb4 in MS5 spectra of all molecular adduct ions found in the precursor ion map, consistent with the presence of both iGb4 and Gb4 isobars in tetraglycosylceramides from human thymus (Figure 3 right panel, and supplementary Figure 1). Supplementary Table I lists the parent ion profiles and results of MS5 analysis. An extremely heterogeneous mixture of iGb4/Gb4 isobars was found for human thymic tetraglycosylceramides. This is ascribed to different length and functionalization of both the fatty acid chains and sphingoids.
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Discussion |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Here we present a first demonstration of the presence of iGb4 in human thymus. In combination with our previous finding that iGb3 is a minor component of human thymic trihexosylceramide (Teneberg et al., in preparation), the results demonstrate that the isoglobo series of GSLs are expressed in human thymus.
Our data contradict a popular hypothesis that human do not express iGb3 (Milland et al. 2005; Milland et al. 2006; Sandrin 2007;Speak et al. 2007). This notion stems from the belief that iGb3 would bind to anti--Gal antibodies, which are naturally expressed in humans and, together with the Gal3Galβ4GlcNAc epitope, would cause hyper acute rejection in xeno-transplantation. Human natural anti--Gal antibodies could bind to synthetic Gal3Galβ4Glc glycan conjugated to BSA in an ELISA assay (Andreana et al. 2004). However, they did not bind to intact iGb3 glycolipid in a TLC-immunoblot, as we previously reported (Teneberg et al. 1996; Zhou et al. 2004). Consistent with our finding, later studies found that organs from 3GalT1 knockout pigs lacking the Gal3Galβ4GlcNAc epitope did not cause hyper acute rejection (Yamada et al. 2005). Nevertheless, it has been suggested that iGb3 expressed in the xenograft would activate NKT cells and ultimately promote rejection (Sandrin 2007). This hypothesis was supported by the failure to detect iGb3 in human tissue sections by immunohistochemistry (Milland et al. 2006), which implied that humans would see and respond to iGb3 as a foreign antigen. It was also suggested that human iGb3 synthase is a pseudogene based on negative finding by reverse-transcription–PCR (RT–PCR) experiments (Milland et al. 2006; Speak et al. 2007). In contrast, we have found the presence of mRNA transcripts for iGb3 synthase in human thymus, and have cloned a full-length cDNA (unpublished data). The detailed enzymatic basis for isoglobo series GSLs in human and in mouse, as well as their regulation, is currently being investigated.
Another group reported the failure to detect iGb3 and iGb4 in human tissues, using an HPLC method assaying fluorescence-labeled glycans cleaved from ceramides (Speak et al. 2007). The same method did not detect iGb3 and iGb4 in the mouse thymus either. Using a similar ion trap MSn-based method as reported here, we demonstrated the presence of iGb3 in both human and mouse thymus (Teneberg et al., in preparation). The discrepancy between these reports may be due to the sensitivity of the different methods. We found that thymic trihexosylceramides (iGb3 and Gb3) were very heterogeneous with respect to their fatty acid components, and overall iGb3 represented a minor fraction of the trihexosylceramides. Speak et al. reported that their HPLC method could detect as little as 1% of iGb3 in an iGb3/Gb3 mixture (Speak et al. 2007). According to our ion trap MSn assay, which distinguishes between trihexosylceramide species that differ by their fatty acid components, many trihexosylceramide species contained less than 1% iGb3, suggest- ing an explanation for the failure of Speak et al. to detect it.
We have successfully established a method to detect iGb4 in biological samples, by comparing the signature MS5 ion fragments to those of the standard iGb4. The selectivity of our method is based on exploiting the ability of the ion trap to (i) specifically isolate the internal HO-3Gal3/4Gal-1-ene disaccharide fragment, containing the iGb4/Gb4 structure-defining isomeric linkages, from each molecular precursor whose m/z and intermediate fragments are consistent with the correct glycan formula and sequence; and (ii) fragment this disaccharide in an additional CID-MS step to yield product ions that distinguish the two linkages, including cross-ring cleavage fragments and further products which are linkage specific (Ashline et al. 2005). Differences in relative abundances of some common fragments can provide additional criteria for confirming the target linkages or ruling out others. Significantly, the method is capable of detecting one isomer in the presence of the other, even where the latter occurs at far higher levels. Where very low levels of GSLs are being analyzed, a precursor ion mapping routine can help to locate candidate molecular ions, even in noisy profile spectra. This was facilitated by the low sample consumption of the nanoelectrospray source. Although precursor ion scanning, as carried out on a tandem MS/CID-MS instrument, is a more efficient and sensitive process for this purpose, tandem instruments cannot perform the required number of fragmentation steps (MS5 in this case) to isolate specific disaccharides and produce linkage-specific fragments from them. A hybrid tandem configuration, such as in a QqLIT/Q instrument, for example, could provide efficient implementation of all the necessary capabilities for higher throughput analysis, if sufficient sensitivity can be maintained.
A further step of development would be to establish a quantitative approach, e.g., by mixing our samples with a known amount of a suitably distinct iGb4 internal standard. These approaches, combined with our methods to measure other intermediate metabolites of the isoglobo series of GSLs (lactosylceramide and glucosylceramide), may help to understand the metabolism of iGb3, which remains the only well-established natural ligand of NKT cells as confirmed by independent laboratories. The ion trap MSn technology described here therefore represents a powerful, generally applicable method for the analysis of other biologically active GSLs, as well as their metabolic pathways.
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Materials and methods |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Tissues and cultured cells
Human thymi from pediatric patients (aged between 1 month to 5 years old) undergoing open-heart surgery were collected and pooled. All human samples were collected under institutionally approved protocols at University of Chicago Hospitals or the University of Texas M.D. Anderson Cancer Center at Houston. The CHO cells and CHO-iGb3 cells have been cultured and harvested as described (Teneberg et al., in preparation), and stored in 16 x 100 mm glass tubes at –80°C.
Per-N,O-methylation of neutral GSLs
A modification of the method of Ciukanu and Kerek (Ciucanu and Kerek 1984; Ciucanu and Costello 2003) was employed for per-N,O-methylation of GSLs. GSLs (1–20 µg) were introduced into a conical glass vial, and dimethyl sulfoxide (150 µL) was added without using special drying conditions or inert gas atmosphere. Powdered sodium hydroxide (40–60 mg) was then added to the sample solution, and was stirred at room temperature until completely dissolved. Iodomethane (80 µL) was added with a syringe, and the mixture was shaked at room temperature for 1 h. The methylation reaction was quenched with water (2 mL). The permethylated products were extracted three times by addition of dichloromethane (2 mL). The combined dichloromethane extracts were then washed three times with water (2 mL each). Following the final wash, the samples were transferred to a new tube, and dried under nitrogen stream at 35–40°C.
Electrospray ionization linear ion trap mass spectrometry (ESI-LIT-MSn) of permethylated neutral GSLs
Electrospray ionization mass spectrometry (MS and MSn) was carried out in positive ion mode on a linear ion trap mass spectrometer (LTQ, ThermoFinnigan, San Jose, CA), using a nanoelectrospray source, with a flow rate of 0.30 µL/min and at capillary temperature 230°C with injection time 100.00 ms, activation time 30 ms, activation Q-value 0.250, isolation width m/z 1.5, and acquisition time 3 min. Normalized collision energies were set to leave a minimal residual abundance of precursor ion; in this case, 30% was used for all product ion scans. All ions were detected as sodium adducts. To obtain MS5 spectra specifically from the characteristic internal HO-3Gal3/4Gal-1-ene disaccharides, each iGb4/Gb4 molecular species (or lipoform, observed at, e.g., m/z X), was subjected to multistep fragmentation via the MS5 pathway X912690431 (corresponding to Scheme 1).
Mapping of precursors of the m/z 912 (912.5) MS2 fragment was performed with a standard subroutine in the ThermoFinnigan Xcalibur software package, using the following parameters: scan filter MS2; activation type, CID; precursor m/z range 1450.0–1650.0, precursor ion step m/z 0.2, isolation width m/z 2.0 Th, product m/z 912.5, normalized collision energy 35%, activation Q-value 0.250, activation time 30 ms, and total run time 100 min. Profiles of iGb4/Gb4 molecular adduct ions were obtained as a "map" of all precursors yielding the MS2 ion at m/z 912.5 (see Figure 3). Each precursor of m/z 912.5 observed in significant abundance (m/z X) was then subjected to MS5 analysis by the X912690431 pathway as described above.
Isobaric iGb4 and Gb4 reference standards
Purified isobaric iGb4 and Gb4 (C70H130N2O23, MW 1366; permethylated MW 1548) were obtained from nonacid GSL fractions by standard procedures as previously described (Teneberg et al. 1996). The individual GSLs were isolated by repeated chromatography of native or acetylated fractions on silicic acid columns and by HPLC. They were stored in chloroform–methanol 1:1 (v/v), and further diluted to obtain standards of concentration as indicated. Both of them (5 µg each) were permethylated and subjected to ESI-LIT-MSn analysis as described above.
Total lipid extraction from CHO cells
Lipids were extracted by extensive sonication four times with mixed polarity solvents. The first and last solvent used was chloroform–methanol 1:1 (v/v). The second and third solvent used was isopropanol–hexane–water 55:25:20 (v/v/v, upper phase removed by aspiration before use). Sonication was followed by centrifugation to pellet the insoluble material. The supernatants were pooled and dried under nitrogen stream at 40°C, and subjected to preliminary analysis by high-performance thin layer chromatography (HPTLC).
Separation of neutral and acidic lipids from CHO cells
Neutral and acidic lipids were fractionated by anion exchange chromatography on a small column of DEAE Sephadex A-25 (Yu and Ledeen 1972) in chloroform–methanol–water 30:60:8 (v/v/v); neutral lipids were eluted with five column volumes of this solvent, while the acidic lipid fraction was eluted with 0.8 M sodium acetate in methanol. Both neutral and acidic fraction were dried, desalted by dialysis, dried by rotary evaporation, and analyzed by HPTLC.
Florisil fractionation of neutral GSLs from CHO cells
The method of Saito and Hakomori (1971) was employed for removing non-GSL impurities from the neutral GSL fraction. The DEAE Sephadex A-25 pass-through fraction was dried under vacuum in the presence of P2O5 for 3 h and peracetylated with 4 mL pyridine and 2 mL acetic anhydride in the dark at room temperature overnight. The peracetylated material was dried by rotary evaporation, with the addition of 2 mL toluene for three times to ensure complete evaporation. A Florisil column (30–60 mesh, 10 x 80 mm) was equilibrated in 1,2-dichloroethane–hexane 4:1 (v/v). The peracetylated sample was applied in this solvent, and the column was then washed with 100 mL of the same solvent, followed by 100 mL 1,2-dichloroethane. Neutral peracetylated GSLs were eluted with 200 mL 1,2-dichloroethane–acetone 1:1 (v/v). The fractions were dried by rotary evaporation and deacetylated with 5 mL 0.5 M sodium methoxide in 10 mL methanol for 3 h at room temperature. The mixture was neutralized with methanolic acetic acid, dried by rotary evaporation, and then desalted by dialysis. The GSL contents of all three fractions were compared by HPTLC; GSLs were always observed to be in the third fraction.
Detection of iGb4/Gb4 tetraglycosylceramides in CHO cells and CHO transfected by iGb3 synthase
Neutral GSLs from CHO cells and CHO-iGb3 cells, isolated by Florisil column were permethylated and subjected to ESI-LIT-MSn analysis as described above. The presence of iGb4/Gb4 was confirmed separately in each lipoform by the analysis of MS5 product ion spectra from each precursor as described above (i.e., X912690431), and relative amounts of them were determined by comparing the different ion abundance of each identified molecular species in the two kinds of CHO cells represented in the MS1 profile spectrum. MS2 spectra were systematically acquired for every molecular precursor ion m/z 912 in the MS1 spectrum from m/z 1450 to 1650 (covering the range of tetraglycosylceramides with ceramide compositions having d18:1, d18:0, or t18:0 sphingoids N-acylated with C16 to C28 fatty acid).
Preparation of tetraglycosylceramide fractions of human thymi and detection of iGb4
Pooled human thymi were lyophilized, followed by extraction in two steps with chloroform and methanol (2:1 and 1:9, v/v), in a Soxhlet apparatus. The extracted material was pooled and subjected to mild alkaline hydrolysis, followed by dialysis and purification on a silicic acid column. Thereafter, acid and nonacid GSLs were separated on a DEAE cellulose column. The nonacid fraction was acetylated and separated from alkali-stable phospholipids on a second silicic acid column. After deacetylation, final purification of the nonacid GSLs was done on DEAE cellulose and silicic acid columns. Aliquots of 200–500 µg were saved at each preparative step. The total nonacid fraction was separated on a 10-g Iatrobeads column (Iatron Laboratories Inc., Tokyo, Japan) by eluting with chloroform/methanol/water (65:25:4, by volume; 20 fractions of 5 mL each). The separation was monitored by thin layer chromatography and anisaldehyde staining (Karlsson 1987).
A mixture of tetraglycosylceramides (10 µg) prepared in this way from human thymus was permethylated as described above. A profile of Gb4 and iGb4 lipoforms was obtained by mapping all precursors of the glycan fragment m/z 912.5; each m/z 912.5 precursor observed in significant abundance was then subjected to MS5 analysis as described above. The iGb4 in the isobaric mixture of Gb4 and iGb4 was identified by comparing the different patterns of MS5 product ions, compared with those of pure permethylated Gb4 and iGb4 standards, as described in the Results section.
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Supplementary Data |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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Supplementary data for this article is available online at [Journal URL].
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Funding |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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S T was funded by Swedish Medical Research Council (Grant No. 12628) and the Swedish Cancer Foundation. A B is an Howard Hughes Medical Institute Investigator. A B is being supported by NIH grant PO1 AI053725. S B L was funded by a grant from NIH/NCRR (R21 RR20355). D Z is funded by MD Anderson Cancer Center and Joe Moakley Leukemia SPORE Developmental Research Project Award from NCI (1 P50 CA 100632-04).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank Dr. Vernon Reinhold and University of New Hampshire Glycomics Center (supported by NIH/NCRR grant P20 RR16459) for providing MS core facility.
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Abbreviations |
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3GalT1, -1,3-glycoprotein galactosyltransfererase 1; ESI-LIT-MS, electrospray ionization linear ion trap mass spectrometry; Gb4, globotetraglycosylceramide; Gb3, globotrihexosylceramide; GSL, glycosphingolipid; HPTLC, high-performance thin layer chromatography iGb4, isoglobotetraglycosylceramide; iGb3, isoglobotrihexosylceramide; LIT, linear ion trap; MS, mass spectrometry; NKT, natural killer T cells; TCR, T cell receptor |
References |
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TopAbstractIntroductionResultsDetection of iGb4 and...DiscussionMaterials and methodsSupplementary DataFundingConflict of interest statementReferences |
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