Glycobiology Advance Access originally published online on July 13, 2005
Glycobiology 2005 15(12):1286-1301; doi:10.1093/glycob/cwj011
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Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway
2 Department of Biology, Georgia Institute of Technology, 309 Cherry Emerson Building, Atlanta, GA 30332-0230; 3 Faculty of Health Sciences, Department of Medical Biochemistry and Genetics, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen N, Denmark; and 4 Department of Chemistry, University of New Hampshire, Durham, NH 03824
1 To whom correspondence should be addressed; e-mail: slevery{at}cisunix.unh.edu
Received on March 28, 2005; revised on July 6, 2005; accepted on July 8, 2005
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Abstract |
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The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcß1Cer
Manß4Glcß1Cer [MacCer] GlcNAcß3Manß4Glcß1Cer [At3Cer] GalNAcß4- GlcNAcß3Manß4Glcß1Cer [At4Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galß3Manß4Glcß1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAc
4Galß3Manß4- Glcß1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAc4 and Galß3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcß4 in the arthro-series penta- and hexaglycosylceramide structures (At5Cer and At6Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAc4 and Galß3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galß3-T capable of using Manß4Glcß1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate. Key words: electrospray ionization / glycolipid / insect / mass spectrometry / NMR spectroscopy
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Glycosphingolipids (GSLs) of insects, as represented by a variety of dipteran species (Sugita et al., 1982a
,b; 1990; Dennis et al., 1985a,b; Dabrowski et al., 1990; Weske et al., 1990; Helling et al., 1991; Itonori et al., 1991; Seppo et al., 2000), differ considerably from those of mammals and other animals in the chordate lineage but share key core glycan features at least through the tetraglycosylceramide At4Cer with nematodes (Gerdt et al., 1997, 1999; Lochnit et al., 1997, 1998, 2001; Wuhrer et al., 2000). The insect pathway diverges from the mammalian starting with the addition of the second glycosyl residue to glucosylceramide (GlcCer) and results in the expression of characteristic "arthro-" (At)-series GSLs: Glcß1Cer [GlcCer] Manß4Glcß1Cer [MacCer] GlcNAcß3Manß4Glcß1Cer [At3Cer] GalNAcß4GlcNAcß3Manß4Glcß1Cer [At4Cer].
Despite the apparently strong resemblance between the dipteran and nematode pathways, independent investigations with the model fly, Drosophila melanogaster, and the model worm, Caenorhabditis elegans, suggest that they are not functionally equivalent in both species. Disruptions of either the Manß4-T or the GlcNAcß3-T in the D. melanogaster pathway are related to phenotypes, egghead and brainiac, respectively (Muller et al., 2002; Schwientek et al., 2002; Wandall et al., 2003), exhibiting obvious developmental defects resulting in severely compromised viability and reproductive capacity (Goode et al., 1992, 1996); the consequences of disrupting the homologous C. elegans genes, on the other hand, appear to be confined primarily to loss of susceptibility of the organism to cryotoxins, the binding of which requires peripheral glycosyl residues carried on the arthro-series tetraglycosylceramide core (Griffitts et al., 2001, 2003, 2005).
During our studies of the genetic regulation of insect glycosylation pathways, Sf9 and High Five insect cells were used for recombinant overexpression of putative Drosophila glycosyltransferases, including the ß4Man-T egghead and the ß3GlcNAc-T brainiac, which operate consecutively in the formation of the arthro-series core glycan. Infection of High Five cells with baculovirus expressing a full coding construct of brainiac resulted in alteration of the GSL profile, as observed by high performance thin layer chromatography (HPTLC) analysis (Schwientek et al., 2002). However, the results were more complex than expected, as there were apparently two separate components in High Five cells with Rf-values similar to triglycosylceramides, whose relative proportions were altered by infection with brainiac. Further analysis revealed that wild-type High Five cells already contained two triglycosylceramide components, a major, low Rf species possessing a novel structure, and a minor, higher Rf species, the abundance of which increased upon infection by brainiac. The latter proved to be the expected brainiac biosynthetic product, GlcNAcß3Manß4Glcß1Cer (GlcNAcß3-MacCer), but full interpretation of the experiment required characterization of the unknown High Five cell component. Preliminary characterization suggested this component was Galß4-MacCer (Schwientek et al., 2002), but more detailed analysis has now been carried out, revealing instead an alternate GSL pathway in High Five cells initiated by addition of Galß3 to MacCer. The elucidations of the major neutral High Five GSLs, primarily by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS), are reported in this article, and the results are discussed with respect to the usual arthro-series pathway and its possible functional significance.
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Results |
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Detection and isolation of High Five GSLs
Three major GSL bands, as well as some lower Rf minor components, were detected by HPTLC analysis with orcinol staining of the crude High Five lipid extract (Figure 1, lane 1). These migrated approximately as di-, tri-, and tetraglycosylceramides (marked MacCer, Hi5-3, and Hi5-4 in Figure 1), compared with a standard containing the mammalian neutral GSLs globotri- and globotetraosylceramide (Figure 1, lane S; Gb3 and Gb4, respectively). Following removal of other lipids, such as cholesterol and phosphodiglycerides, a similar profile was obtained (Figure 1, lane 2). This material was fractionated by high performance liquid chromatography (HPLC), and purified samples of the three major components were subjected to structural analysis by NMR and MS methods.
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NMR spectroscopic analysis of major High Five GSLs
The downfield regions of one-dimensional 1H-NMR spectra of three purified fractions are reproduced in Figure 2, along with the deduced glycan sequences. The glycan sequences of the novel components Hi5-3 and Hi5-4 were completely elucidated by analysis of homonuclear and heteronuclear two-dimensional NMR experiments. The process typically included acquisition of (1) 1H-1H-gCOSY and series of 1H-1H-TOCSY experiments with increasing mixing times (30–200 msec) for assignments of spin system connectivities and 1H resonance chemical shifts, followed by monosaccharide residue configurational identification using relative stereochemical assignments extracted from the magnitudes of three-bond scalar coupling constants between vicinal sugar ring protons (the 3Ji,j-coupling magnitudes need only be approximately determined; Koerner et al., 1987; Dabrowski, 1989); (2) 1H-detected 1H-13C-gHSQC spectra for 13C chemical shift assignments via one-bond 1H-13C connectivities; and (3) 1H-1H-NOESY and/or 1H-detected 1H-13C-gHMBC experiments for the determination of glycosidic linkages—via strong through-space dipolar cross relaxations originating from each sugar H-1 in the former case and three-bond interglycosidic 1H-13C correlations in the latter. Salient points of the NMR spectral analyses are the following.
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Diglycosylceramide (MacCer)
The one-dimensional 1H-NMR spectrum of the diglycosylceramide component (Figure 2A) exhibited two H-1 resonances with chemical shifts and 3J1,2 splitting consistent with ß-Manp (4.498 ppm; 3J1,2 
1 Hz) and ß-Glcp (4.156 ppm; 3J1,2 = 7.8 Hz) residues in Manß4Glcß1Cer; this was confirmed by assignment of spin system connectivities from two-dimensional 1H-1H correlation NMR experiments and sugar ring stereochemistries from approximate magnitudes of the respective sets of vicinal three-bond coupling constants. J connectivities, coupling magnitudes, and chemical shift patterns for ceramide containing an (E)-4-sphingenine base were also apparent in the spectra, including characteristic coupled vinyl H-4 and H-5 multiplets at 5.364 and 5.545 ppm, respectively. The MacCer glycan structure is well known from previous descriptions of GSLs of dipteran larvae and pupae (e.g., Calliphora vicina, Lucilia caeser, D. melanogaster) (Dennis et al., 1985a; Sugita et al., 1982b; Seppo et al., 2000), as well as nematodes (e.g., Ascaris suum, C. elegans) (Gerdt et al., 1997; Lochnit et al., 1997), and it was not subjected to further NMR analysis in this work. 1H chemical shift data for this MacCer component are listed for reference in Table I.
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Triglycosylceramide (Hi5-3)
The one-dimensional 1H- NMR spectrum of the putative triglycosylceramide component (Figure 2B) exhibited three major H-1 resonances with chemical shifts and 3J1,2 splitting consistent with ß-Manp (4.544 ppm; 3J1,2 1 Hz) and ß-Glcp (4.160 ppm; 3J1,2 = 7.8 Hz), with one additional H-1 signal (4.255 ppm; 3J1,2 = 7.6 Hz) suggesting the presence of a third ß-linked hexose residue in the sequence. The downfield shift of the ß-Manp H-1 and lack of significant effect on the ß-Glcp H-1 suggested glycosylation of the Man residue of Manß4Glcß1Cer. The spectrum was clearly distinct from that of the brainiac product GlcNAcß3Manß4Glcß1Cer, in which the ß-GlcpNAc H-1 signal resonates much further downfield, almost coincident with that of the ß-Manp H-1 (Schwientek et al., 2002); moreover, the High Five triglycosylceramide spectrum substantially lacked an upfield NAc methyl group 3H singlet resonance characteristic of a HexNAc residue (note that a low magnitude singlet was observed at 1.829 ppm, with an integrated area <0.5H, along with minor resonances observable in the anomeric region ~4.53–4.51 ppm; see ESI-MS and MS/CID-MS analysis of major High Five GSLs for the discussion of this point). All resonances of the three glycosyl spin systems were assigned straightforwardly by two-dimensional 1H-1H-gCOSY and -TOCSY experiments (Table I), and analysis of the ring proton coupling patterns indicated the third residue to be a ß-Galp, which suggested already a novel, non-arthro-series sequence warranting careful assignment of the new linkage.
Upon assignment of 13C resonances via a two-dimensional 1H-13C-gHSQC spectrum (Figure 3A; Table II), it was apparent that two 13C resonances were shifted significantly downfield, ß-Glcp C-4 and ß-Manp C-3; the latter strongly indicated a Galpß13Manp linkage. Interestingly, all ring 1H resonances for the ß-Manp residue were shifted significantly downfield, with the strongest (and essentially equal effects) observed on both H-2 and H-3 (compare Manß4Glcß1Cer and Galß3Manß4Glcß1Cer in Table I); however, ß-Manp C-2 was not observed downfield (Table II). The Galpß13Manp linkage was unambiguously confirmed by the acquisition of a 1H-13C-gHMBC spectrum (Figure 3A, insets), which displayed clear three-bond inter-residue correlations between ß-Galp H-1 and ß-Manp C-3 and between ß-Galp C-1 and ß-Manp H-3 (in addition to ß-Manp H-1/ß-Glcp C-4, ß-Manp C-1/ß-Glcp H-4; ß-Glcp H-1/Sph C-1; and ß-Glcp C-1/Sph H-1a,1b). The structure Galß3 Manß4Glcß1Cer is therefore proposed with confidence for Hi5-3.
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Tetraglycosylceramide (Hi5-4)
The one-dimensional 1H-NMR spectrum of the putative tetraglycosylceramide component (Figure 2C) exhibited four H-1 resonances with chemical shifts and 3J1,2 splitting consistent with ß-Manp (4.556 ppm; 3J1,2 1 Hz) and ß-Glcp (4.159 ppm; 3J1,2 = 7.8 Hz), with the H-1 signal for ß-Galp in the Galß3Manß4Glcß1Cer substructure shifted somewhat downfield (4.314 ppm; 3J1,2 = 7.0 Hz), and a new H-1 signal (4.828 ppm; 3J1,2 = 3.7 Hz) and an NAc methyl group singlet (1.829 ppm), suggesting the presence of an -linked HexNAc in the sequence, possibly linked to the ß-Galp residue. All resonances of the four glycosyl spin systems were assigned by two-dimensional 1H-1H-gCOSY and -TOCSY experiments (Figure 4A and B; Table I), and analysis of the ring proton coupling patterns indicated the fourth residue to be an -GalpNAc. Assignment of 13C resonances via a two-dimensional 1H-13C-gHSQC spectrum was again fairly straightforward (Table II), and in addition to the downfield shifts previously noted for ß-Glcp C-4 and ß-Manp C-3 in Hi5-3, a significant downfield shift was now observed for ß-Galp C-4 compared with its value in Hi5-3 (
= +7.2 ppm), strongly suggesting a terminal Galp NAc14Galp linkage and overall a GalpNAc4Galß3 Manß4Glcß1Cer tetraglycosylceramide sequence.
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Unfortunately, because of sample limitations, not all of the key three-bond interresidue correlations were observable in a 1H-13C-gHMBC spectrum (not shown); however, useful two-dimensional 1H-1H-NOESY spectra were acquired, supporting the proposed structure for Hi5-4. As shown in Figure 4C, a very strong dipolar cross-relaxation correlation demonstrates the spatial proximity of -GalpNAc H-1 to ß-Galp H-4; no other significant interresidue correlations originate from this proton. Note that in the case of Galp (NAc)13Galp linkages (as commonly found, e.g., in the human blood group A and B determinants), it has been known for some time that the strongest interresidue NOE correlations are observed between -Galp(NAc) H-1 and GalpH-4 (Lemieux et al., 1980; Dua et al., 1986; Rao and Bush, 1988; Yan and Bush, 1990); this situation is brought about by conformational constraints which are predictable from molecular mechanics or dynamics calculations taking the exo-anomeric effect into account (Lemieux et al., 1980; Yan and Bush, 1990). This might appear to render any conclusions from observation of this interaction being somewhat ambiguous. However, as pointed out by Dabrowski (1987, 1989), in the case of a Galp(NAc)13Galp linkage, a significant cross-relaxation is still also observed between Galp(NAc) H-1 and Galp H-3, and the complete absence of such a correlation in the NOESY spectrum of Hi5-4 would almost certainly rule out an 13 linkage, even in the absence of other data, such as the downfield shift of ß-Galp C-4.
The NOE interactions originating from ß-Galp H-1 represent another interesting case, as the intense cross-peak correlating with ß-Manp H-3 is accompanied by another of comparable magnitude with ß-Manp H-2. Again, the potential ambiguity in interpretation of linkage is alleviated by the examination of a molecular model for this disaccharide, which shows that the proximity of ß-Galp H-1 and ß-Manp H-2 is conformationally allowed within the constraints of a Galpß13Manp linkage, whereas the close approach of ß-Galp H-1 to ß-Manp H-3 is essentially ruled out in any conformation of the alternative Galpß12Manp linkage.
ESI-MS and MS/CID-MS analysis of major High Five GSLs
Glycan structures with respect to Hex/HexNAc ratios and sequences were confirmed by +ESI-MS and +ESI-MS/CID-MS analysis; this technique also provided key information on the size distribution of the ceramide fatty-N-acyl and sphingoid (Sph) components and gave evidence for the presence of minor components in some fractions. In these spectra, the observed molecular adduct profiles were consistent with typical dipteran ceramide distributions (Sugita et al., 1982a,b; 1990; Dennis et al., 1985a,b; Weske et al., 1990; Helling et al., 1991; Itonori et al., 1991), that is, having d14:1 and d16:1 sphing-4-enines in combination mainly with saturated 20:0 and 22:0 fatty acids. Saturated 16:0, 18:0, and 24:0 fatty acids have also been detected in these dipteran GSLs; however, GC/MS analysis of the fatty acid methyl esters derived from methanolysis of the High Five GSLs detected only 20:0, 22:0, and smaller amounts of 18:0 species (data not shown).
Diglycosylceramide (MacCer)
In the +ESI-MS molecular ion profile of the MacCer fraction (Figure 5A), a group of presumptive sodiated molecular ions at m/z 856, 884, 912, and 940 is accompanied by another equally abundant set, differing systematically by 16 u from the first, at m/z 872, 900, 928, and 956. At this low level of m/z resolution and accuracy, the latter may commonly be attributed to either K+ adducts or the presence of molecular species with hydroxylated fatty-N-acylated ceramide components, but the latter would be inconsistent with the GC/MS analysis, in which no hydroxy fatty acids were detected. Following the addition of LiI (Figure 5B), lithiated molecular adducts were detected mainly at m/z 840, 868, 896, and 924, consistent with Hex2Cer with 18:0/d14:1, 18:0/d16:1 + 20:0/d14:1, 20:0/d16:1 + 22:0/d14:1, and 22:0/d16:1 ceramides; residual sodiated adducts are also observed in the profile at low abundance. This confirms that the second set of ions in the original profile corresponds to K+ adducts. An additional set of low abundance ions appears in the Li+ adduct profile (Figure 5B), consisting of species 6 u higher than the major lithiated species, for example, at m/z 874 and 902. Because no corresponding ions were observed in the native profile (Figure 5A), these appear to be artifacts, possibly resulting from net replacement of a hydroxyl proton with a second lithium ion. These species were also observable in the lithium adduct profiles of later fractions but were not investigated further for this study, as their occurrence did not affect any of the structural conclusions.
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The deduced ceramide features were further confirmed by the acquisition of product ion +ESI-MS/CID-MS of selected molecular species (Figure 6A–C). Sph/fatty-N-acyl distributions were reflected in the observation of N'' (non-lithiated double dehydration of Sph Y0/O; Scheme 1) ions at m/z 208 and/or 236 for the d14:1 and/or d16:1 species, respectively (note the appearance of both in the tandem spectrum of the m/z 896 species [Figure 6B]). Corresponding T ions (Z0/G; Scheme 1) including the fatty-N-acyl chain are observed at m/z 344 and 372 for the 20:0 and 22:0 species, respectively (note the appearance of both in the tandem spectrum of the m/z 896 species [Figure 6B]).
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Glycan sequences were confirmed in particular by the observation of abundant Y-series ions (Scheme 1) in the tandem +ESI-MS/CID-MS product ion spectra (Figure 6A–C), as well as B- and C-series ions, although these latter fragments are not always possible to distinguish from internal B/Y and C/Y ions. For example, for the m/z 868 species of MacCer (Figure 6A), Y1 and Y0 fragments are found at m/z 706 and 544, respectively.
Triglycosylceramide (Hi5-3)
Two different HPLC fractions of triglycosylceramide gave somewhat different Li+ adduct profiles, as shown in Figure 7A and B. Most peaks observed overall were consistent with Li+ adducts of a Hex3Cer with 18:0/d14:1, 18:0/d16:1 + 20:0/d14:1, 20:0/d16:1 + 22:0/d14:1, and 22:0/d16:1 ceramides (m/z 1002, 1030, 1058, 1086, accompanied by residual Na+ adducts at m/z 1018, 1046, 1074, and 1102, respectively). The major differences were consistent with variations in ceramide size distribution. Interestingly, however, peaks with odd-numbered m/z ratios, 1071 and 1099, appeared with variable abundance in both spectra, consistent with a general composition of HexNAc1Hex2Cer. Sequence differences corresponding to the apparent substitution were reflected in the corresponding tandem +ESI-MS/CID-MS product ion spectra (Figure 8A–C). The major molecular components, for example, m/z 1030 and 1058, yielded sequential Yn product ions, all differing by m/z 162, consistent with the linear Galß3Manß4Glcß1Cer (Hi5-3) structure (Figure 8A, m/z 868, 706, 544; Figure 8B, m/z 896, 734, 572); the minor components, for example, m/z 1099, yielded sequential Yn product ions differing by m/z 203, 162, and 162, consistent with a linear HexNAc-O-Hex-O-Hex-O-Cer sequence (Figure 8C, m/z 896, 734, 572), as found in GlcNAcß3Manß4Glcß1Cer (At3Cer). Close inspection of the one-dimensional 1H-NMR spectrum of this fraction (Figure 2B) confirms the identity of this minor component, the triglycosylceramide intermediate normally observed in the dipteran (and nematode) GSL biosynthetic pathway. As mentioned earlier, its presence is consistent with the minor resonances observed at 1.829 ppm (GlcNAc NAc methyl singlet) and 4.528 (ß-Manp H-1, 3J1,2 1 Hz), along with an apparent downfield shoulder on the ß-Glcp H-1 resonance which would indicate a minor ß-Glcp H-1 shifted to ~4.17 ppm.
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Tetraglycosylceramide (Hi5-4)
Peaks observed in the +ESI-MS profile at m/z 1205, 1233, 1261, and 1289 (Figure 9A) were consistent with Li+ adducts of a HexNAc1Hex3Cer having a ceramide distribution similar to those discussed previously. In the tandem +ESI-MS/CID-MS product ion spectra of the major components (Figure 9B and C), m/z 1233 and 1261, sequential Yn product ions differing by m/z 203, 162, 162, and 162 were observed, consistent with the linear HexNAc-O-Hex-O-Hex-O-Hex-O-Cer sequence corresponding to GalNAc
4Galß3Manß4Glcß1Cer (Figure 9B, m/z 1030, 868, 706, 544; Figure 9C, m/z 1058, 896, 734, 572). Although some of the Yn fragments are observed at very limited abundance in these low energy CID spectra (m/z 868 in particular), those corresponding to the initial HexNAc loss (m/z 1030 and 1058) are unmistakable and sufficient to confirm its nonreducing terminal position in the sequence.
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Glycosyl composition and linkage analysis
The identity of all monosaccharide residues comprising MacCer, Hi5-3, and Hi5-4 was confirmed by gas chromatography/mass spectrometry (GC/MS) analysis of per-O-trimethylsilyl derivatives of the methyl glycosides produced by acidic methanolysis of the GSLs (data not shown). Confirmation of all proposed linkages was provided by methylation analysis via GC/MS of partially methylated alditol acetate (PMAA) derivatives (data not shown). Following permethylation, depolymerization, reduction, and peracetylation of MacCer, the expected PMAA derivatives corresponding to T-Man (2,3,4,6-tetra-O-Me-Man) and
4Glc (2,3,6-tri-O-Me-Glc) were detected. The linkage structure of Hi5-3 was confirmed by the detection of PMAAs corresponding to T-Gal (2,3,4,6-tetra-O-Me-Gal) and 3Man (2,4,6-tri-O-Me-Man), consistent with the proposed new linkage, along with 4Glc (2,3,6-tri-O-Me-Glc). For Hi5-4, PMAAs were detected corresponding to T-GalNAc (3,4,6-tri-O-Me-GalNAcMe) and 4Gal (2,3,6-tri-O-Me-Gal), confirming the new linkage, along with 3Man (2,4,6-tri-O-Me-Man) and 4Glc (2,3,6-tri-O-Me-Glc). |
Discussion |
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GSLs of insects, as exemplified by dipteran species, were extensively characterized throughout the 1980s and early 1990s (reviewed by Dennis and Wiegandt, 1993
). Subsequently, structures of GSLs in the model dipteran, D. melanogaster, were also characterized (Seppo et al., 2000), and many glycosyltransferases in the pathway for their biosynthesis have been identified and characterized at the gene and gene product level (Muller et al., 2002; Schwientek et al., 2002; Wandall et al., 2003; Kohyama-Koganeya et al., 2004; Mucha et al., 2004) (Scheme 2, pathway A). In particular, the Drosophila neurogenic genes egghead and brainiac (Goode et al., 1992, 1996; Goode and Perrimon, 1997) were shown to encode the ß4Man-T and ß3GlcNAc-T required for the biosynthesis of GlcNAcß3Manß4Glcß1Cer (At3Cer), and expression of this pathway, at least through At3Cer, is therefore essential for proper development and fertility (Wandall et al., 2005). On the other hand, genes encoding subsequent glycosyltransferases in the pathway, producing At4Cer (Haines and Irvine, 2005) and At5Cer (Mucha et al., 2004), may not be as essential.
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It was anticipated that GSLs from High Five cells would reflect expression of the arthro-series pathway. However the studies described herein showed the structure of the predominant High Five triglycosylceramide to be Galß3Manß4Glcß1Cer, whereas the predominant tetraglycosylceramide was GalNAc4Galß3Manß4Glcß1Cer (Scheme 2, pathway B). The expected arthro-series triglycosylceramide, GlcNAcß3Manß4Glcß1Cer (At3Cer), was present, but only as a minor component. It should be noted that a significant fraction of dipteran GSLs incorporate a phosphoethanolamine (PEtn) group at the 6-position of the GlcNAcß3 residue (Dennis and Wiegandt, 1993; Seppo et al., 2000), a modification for which there is no acceptor substrate in the dominant High Five pathway.
Available insect genomes indicate the existence of multiple putative ß3-glycosyltransferase genes, and several Galß3 linkages are found in GSLs of diptera, but this residue is normally added in a later part of the dipteran GSL glycan sequence. To the best of our knowledge, the Galpß13Manp-R linkage has not been previously observed in GSLs of insects or any other animal species, but these results imply that at least one insect Galß3-T is also capable of using Manß4Glcß1Cer as an acceptor substrate. The GalNAc4 unit is also found in insect GSLs, but always as the fifth residue linked to GalNAcß4 in the arthro-series pentaglycosylceramide [At5Cer] structure (Scheme 2, pathway A). The terminal disaccharide sequence of Hi5-4 thus reflects a reversal of the normal order of addition in the arthro-series pathway, which yields a terminal Galß3GalNAc4 in the dominant At6Cer structure (Dennis et al., 1985b; Sugita et al., 1990; Helling et al., 1991; Itonori et al., 1991; Seppo et al., 2000).
The existence of an alternate core trisaccharide in High Five GSLs implies that at least in this cell line there is potential for the generation of multiple pathways, with a possibility for core switching as observed in mammalian cells. That Hi5-3 is itself a substrate for further glycosylation by 4GalNAc-T, the existence of which in the dipteran repertoire has already been demonstrated at both the gene/gene product (Mucha et al., 2004) and GSL product (Dennis and Wiegandt, 1993; Seppo et al., 2000) levels is perhaps a remarkable happenstance, but supports the premise that the potential for sequence variability is inherent in the nature of the machinery for glycosylation. On the other hand, the opportunities for this potential to manifest itself must be more limited in a complex, developing organism than in a cultured cell line where the expression of a specific set of GSL structures may be far less critical. For example, the potential existence of a Galß3Manß4Glcß1Cer core pathway is not sufficient to rescue the brainiac mutation, either because its synthesis is prevented by any number of other factors or because its expression is even less viable than brainiac in that context. Nevertheless, the observation that brainiac is essential for development does not rule out the existence of one or more alternative glycosylation pathways operating during the life cycle of a normal fly, that could be revealed by further, more detailed studies of dipteran GSL expression.
It is worth noting, moreover, that studies of GSLs of various fresh- and saltwater bivalve species have indicated already that many alternate possibilities for glycosylation of Manß4 of MacCer exist; such species produce an assortment of GSL glycan structures based on Man3Manß4Glcß1Cer and Man3(Xylß2)Manß4Glcß1Cer core structures (Itasaka and Hori, 1979; Sugita et al., 1981, 1984, 1985; Hori et al., 1983; Itasaka et al., 1983; Scarsdale et al., 1986). Various other structures have also been reported sporadically from bivalves, including Man4Manß4Glcß1Cer, Manß4Manß4Glcß1Cer, and Gal3Manß4Glcß1Cer (Itasaka et al., 1976; Hori et al., 1977a,b; Sugita et al., 1985). Thus, the failure of Galß3-T to rescue brainiac does not preclude the possibility that such an alternative pathway could arise and be found usable in another lineage. One further caveat to be considered here is that High Five cells originate from Trichoplasia ni, a Lepidopteran species, GSLS of which have never been characterized.
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Materials and methods |
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Solvents for GSL extraction and HPTLC analysis
Solvent A, 2-propanol–hexane–water (55:25:20, v/v/v, upper phase discarded); solvent B, chloroform–methanol (1:1, v/v); solvent C, chloroform–methanol–water (60:40:9, v/v/v + 0.03% CaCl2); and solvent D, 2-propanol–hexane–water (55:40:5, v/v/v).
Extraction and purification of High Five cell GSLs
High Five cells were grown in shaking upright roller bottles at 27°C in serum-free medium (Invitrogen, Carlsbad, CA). Briefly, GSLs were extracted by homogenizing High Five cells (~50 mL packed cells) two times with 200 mL of solvent A and two times with 200 mL of solvent B. The four extracts were pooled, dried on a rotary evaporator, and peracetylated by the addition of 100 mL pyridine and 50 mL acetic anhydride, sonication, and reaction overnight in a sealed vessel at room temperature. Following removal of reagents, the peracetylated mixture was subjected to a stepwise chromatography on Florisil, according to the procedure of Saito and Hakomori (1971) to remove lipids (i.e., cholesterol, phospholipids) other than GSLs. Following de-O-acetylation, the GSLs were fractionated by preparative scale HPLC on a 50 x 0.46 cm column packed with 10-µm porous spherical silica (Sphereclone; Phenomenex, Torrance, CA). Similar to previously developed procedures (Kannagi et al., 1987; Levery et al., 1989), the mobile phase was a 2-propanol–hexane–water gradient programmed from 55:40:5 to 55:25:20 over 120 min, followed by isocratic elution for 40 min; flow rate 0.5 mL/min. Fractions were surveyed by analytical HPTLC, with pure fractions pooled according to migration behavior and subjected to analysis by NMR spectroscopy and mass spectrometry as described below.
HPTLC
HPTLC was performed on silica gel 60 plates (E. Merck, Darmstadt, Germany) using solvent C as mobile phase. Neutral lipid samples were dissolved in solvent D and applied by streaking from 5-µL Micro-caps (Drummond, Broomall, PA). Detection was made by Bial’s orcinol reagent (orcinol 0.55% [w/v] and H2SO4 5.5% [v/v] in ethanol/water 9:1 [v/v]; the plate is sprayed and heated briefly from ~200 to 250°C).
Characterization of GSL fractions by NMR spectroscopy
Samples of underivatized GSL (~0.5–1.0 mg) were deuterium exchanged by repeated evaporation from CDCl3/CD3OD (2:1 v/v) under N2 stream at 37°C and then dissolved in 0.5 mL dimethylsulfoxide (DMSO)-d6/2% D2O (Dabrowski et al., 1980) for NMR analysis. One- and two-dimensional 1H-NMR spectra were acquired at 35°C on Varian Unity Inova 600 or 800 MHz spectrometers (University of Georgia/Complex Carbohydrate Research Center, Athens, GA). Two-dimensional 1H-1H-gCOSY, -TOCSY, -NOESY, and 1H-detected 1H-13C-gHSQC and -HMBC experiments were performed using standard acquisition software available in the Varian VNMR package. Proton chemical shifts are referenced to internal tetramethylsilane ( = 0.000 ppm) and carbon chemical shifts to the natural abundance 13C methyl resonance of solvent DMSO-d6 ( = 40.0 ppm).
Characterization of GSL fractions by +ESI-MS and tandem MS/CID-MS
Positive ion ESI-MS was performed on a PE-Sciex (Concord, Ontario, Canada) API-III spectrometer equipped with a standard IonSpray source (University of Georgia/Complex Carbohydrate Research Center). Procedures and conditions for +ESI-MS and tandem +ESI-MS/CID-MS analysis of GSLs as Li+ adducts were essentially as described previously (Levery et al., 2000, 2001). Briefly, GSL samples were introduced into the ESI source by direct infusion (3–5 µL/min) of solutions in 100% MeOH (analyte concentration 20 ng/µL), to which was added a solution of LiI (10 mM) in MeOH until the observed ratio of Li+ to Na+ molecular adducts in the +ESI-MS profile mode was >90:10 (the final concentration of LiI is generally 2–3 mM for neutral GSLs). Spectra were acquired with orifice-to-skimmer voltage (OR), 90–120 V (low OR), or 150–180 V (high OR); ionspray voltage, 5 kV; interface temperature, 45°C. For +ESI-MS/CID-MS experiments, OR was set to 90–120 V, the collision gas was argon (collision gas temperature [CGT] = 380–400 [x1012 molecules/cm2]), and collision energy was 80 eV. Other parameters were set, as previously described (Levery et al., 2000). Spectra were interpreted as described previously (Levery et al., 2000, 2001).
Glycosyl and fatty-N-acyl component analysis by GC/MS
Glycosyl and fatty acid compositions were obtained by GC/MS analysis after methanolysis of GSLs; the former were detected as per-O-trimethylsilyl methyl glycosides and the latter as methyl esters. Derivatization protocols, GC/MS system, and instrumental conditions for analysis were those described in Levery et al. (1998).
Glycosyl linkage analysis by GC/MS
GSLs were permethylated using the procedure described by Ciukanu and Kerek (1984). Depolymerization of the N,O-permethylated GSLs, reduction, and acetylation to produce PMAAs were carried out according to the protocols described by Levery and Hakomori (1987). PMAAs were analyzed on a Thermo-Finnigan (San Jose, CA) GC/MS system consisting of a Trace Ultra GC interfaced to a Polaris Q ion trap mass spectrometer. Two GC columns were employed: DB-5 ms (Restek, Bellefonte, PA) and DB-210 (J & W Scientific, Rancho Cordova, CA). Both columns were 30 m x 0.25 mm internal diameter, 0.25 µm film thickness, splitless injection, and temperature programmed from 130 to 250°C at 4°C/min. PMAA derivatives were identified by retention times and characteristic EI-IT mass spectra (e.g., Hellerqvist, 1990) compared with those of authentic standards.
Shorthand sequence nomenclature follows the IUPAC recommendations of 1976 and 1997 (IUPAC, 1978; Chester, 1998). The core designation "At-" has been recommended by IUPAC for the arthro-series, replacing "Ap-" (Chester, 1998).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The authors gratefully acknowledge the technical assistance of Sherry Castle for performing the permethylation linkage analysis. This work has been supported by the National Institutes of Health Resource Center for Biomedical Complex Carbohydrates (NIH/NCRR P41 RR05351), the New Hampshire Biological Research Infrastructure Network-Center for Structural Biology (NIH/NCRR P20 RR16459), and the Danish Research Council and the Human Frontier Science Program.
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Abbreviations |
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CID, collision-induced dissociation; DMSO, dimethylsulfoxide; ESI, electrospray ionization; GC/MS, gas chromatography/mass spectrometry; gCOSY, gradient-enhanced correlation spectroscopy; gHSQC, gradient-enhanced heteronuclear single-quantum correlation; gHMBC, gradient-enhanced heteronuclear multiple-bond correlation; GlcCer, ß-glucopyranosylceramide; GSL, glycosphingolipid; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; MacCer, mactosylceramide (Manß4Glcß1Cer); MS, mass spectrometry; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PMAA, partially methylated alditol acetate; Sph, sphingoid; TOCSY, total correlation spectroscopy
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