Unité de Glycobiologie structurale et Fonctionnelle, UMR CNRS no. 8576, Laboratoire de Chimie Biologique, Université des Sciences et Technologies de Lille, F-59655 Villeneuve dAscq, France, 2Unité de Biologie et Physiopathologie des Cellules Mucipares, INSERM U-377, place de Verdun, F-59045 Lille, France, and 3Institut Biomédical des Cordeliers, INSERM U-505, 15 rue de lEcole de Médecine, F-75006 Paris, France
Received on August 10, 1999; revised on December 1, 1999; accepted on December 3, 1999.
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Abstract |
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Key words: aryl-N-acetyl--D galactosaminide/HT-29/oligosaccharide/onco-fetal
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Introduction |
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The addition of aryl-glycosides to the culture medium of cells producing mucins gave rise (as expected) to an increased expression of the Tn antigen (GalNAc(1-)Thr/Ser), but also of the T antigen (Galß13GalNAc(
1-)Ser/Thr), together with a decrease in fucosylated and/or sialylated epitopes (Kuan et al., 1989
; Huang et al., 1992
; Byrd et al., 1995
; Huet et al., 1995
; Delannoy et al., 1996
). In fact, GalNAc
-O-bn is highly converted into the aryl disaccharide Galß13GalNAc
-O-bn, a potent competitive inhibitor of sialyltransferases, fucosyltransferases and N-acetyl-glucosaminyltransferases (Huang et al., 1992
; Delannoy et al., 1996
). In this way, several complex GalNAc
-O-bn derived oligosaccharides were detected in the culture medium and/or cytosol of treated cells after metabolic labeling (Kuan et al., 1989
; Huang et al., 1992
; Byrd et al., 1995
; Delannoy et al., 1996
).
Differentiated mucus-secreting or enterocyte-like populations can be isolated from the human colon carcinoma cell line HT-29 (Lesuffleur et al., 1990; Lesuffleur et al., 1991
). We previously showed that permanent GalNAc
-O-bn treatment of differentiated HT-29 cells leads to marked effects upon the phenotypic properties of mucin-secreting or enterocyte-like HT-29 cells: (1) an inhibition of mucin secretion, (2) a blockade of the apical targeting of membrane brush border glycoproteins such as dipeptidylpeptidase IV, carcinoembryonic antigen and the mucin-like glycoprotein MUC 1, and (3) a swelling of the cells with intracellular accumulation of numerous vesicles (Hennebicq-Reig et al., 1998
; Huet et al., 1998
). Such effects could not be observed after similar treatment of enterocyte-like Caco-2 cells. In order to gain more knowledge about the nature of the material accumulated in these vesicles and on possible modification of the glycolipid metabolism of the GalNAc
-O-bn treated HT-29 cells, we undertook a comparative investigation of the material extracted by organic solvents in control and GalNAc
-O-bn treated enterocyte-like HT-29 and Caco-2 cells. The results showed that treated HT-29 cells specifically accumulate a considerable amount of a series of complex benzylated oligosaccharides that were isolated and structurally characterized.
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Results |
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The compounds accumulated in HT-29 G cells are not glycosphingolipids but GalNAc-O-bn derived oligosaccharides
In order to determine the composition of the compounds accumulated in the GalNAc-O-bn treated HT-29 cells, the material collected in the upper phase was methyl-esterified and analyzed using the MALDI-TOF technique (Figure 4a). In a parallel experiment, an aliquot of the material previously submitted to partial hydrolysis (desialylated) was analyzed (Figure 4b). The deduced masses of the compounds (Table I) indicated the absence of ceramide constituents (this point was checked using GC/MS analysis of the heptafluorobutyrate derivatives obtained after methanolysis) (Zanetta et al., 1999
). In contrast the mass spectra were compatible with derivatives containing GalNAc
-O-bn as common motive. Compounds were first formed by the addition of one hexose or two hexoses, or by the addition of one or two hexoses and one hexosamine. One or two sialic acid residues were present on the largest compounds, which could also contain one deoxy-hexose residue. This was confirmed using the MALDI-TOF spectrum of the desialylated mixture (Figure 4b). Indeed (Table I), the sodium and potassium adducts of desialylated compounds corresponded to GalNAc
-O-bn substituted by one hexose (M = 496), two hexoses (M = 658), one hexose and one N-acetyl-hexosamine (M = 699), two hexoses and one N-acetyl-hexosamine (M = 861), two hexoses, one N-acetyl-hexosamine and one deoxy-hexose (M = 1007).
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[1H]-NMR and methylation studies of the different GalNAca-O-bn derivatives.
The majority of the compounds were analyzed by 1H-NMR, a technique which allowed the assignment of the complete structure of most GalNAc-O-bn derivatives (Table II). This is illustrated in Figure 5 for one of the most complex isolated benzyl derivatives (compound 10). All of the compounds (with the exception of compound 3) possessed the characteristic signals of the proton of the C-2 (2I in Figure 5) of the GalNAc substituted on the C-3. For confirmation of important structures (compounds 2, 3, 6, 9, 10, and 11), methylation analyses were performed, allowing the verification of the different structures reported in Table II and Figure 7. Starting from GalNAc
-O-bn, three families of compounds were identified, corresponding to the addition of further residues on the GalNAc: Galß13 and/or GlcNAcß16 or Neu5Ac
26. The simplest of the orcinol-positive spots (1) was Galß13GalNAc
-O-bn. As shown in Figure 7, the Galß13 branch could be substituted either by another Galß13 residue (2) or by a Neu5Ac
23 residue (9-a, 10, 11). In addition, the GalNAc residue of the Galß13GalNAc
-O-bn motive could also be substituted by a GlcNAcß16 (4). This GlcNAc residue constituted the starting point of a series of compounds, firstly always comprising an additional Gal ß14 (6) that could be sialylated by Neu5Ac
23 (9-b, 10, 11). The GlcNAc residue could be itself substituted by a Fuc
13 (9b and 11), forming the sialyl-Lex motive. Interestingly, compound 3 was unambiguously identified as Galß14GlcNAcß16GalNAc
1-O-benzyl, a member of the core-6 series of O-glycans. Using NMR and methylation experiments, a definite structure could be assigned with reliability to almost all of the benzyl-GalNAc derivatives identified by MALDI-TOF analysis and also discriminating between isomers of the same mass (compounds 3 and 4). This culture system allowed in particular the synthesis of sialyl-Lex derivatives (the Lex derivatives being accumulated at a very low level).
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Analysis of the putative presence of benzylated oligosaccharides in the culture medium
The culture media of Caco-2 cells and of HT-29 G cells were recovered every day over a period of 16 days of treatment with GalNAc-O-bn, lyophilized, and then treated as above. Besides an excess of glucose, two orcinol positive compounds were present in a significant amount in the culture media of treated HT-29 G and Caco-2 cells (data not shown). These compounds showed the same Rf as GalNAc
-O-bn and Galß13GalNAc
-O-bn respectively. The quantity of Galß13GalNAc
-O-bn increased with time in the culture medium of HT-29 G cells, whereas it remained at a lower level in the Caco-2 cell culture medium. In addition, a small amount of the series of complex benzylated oligosaccharides was also detected in the culture medium of HT-29 G cells.
Study of the degradation of benzylated oligosaccharides
As the cellular alterations of HT-29 cells were known to be reversible after the removal of GalNAc-O-bn from the culture medium, we examined the evolution of the synthesized, benzylated oligosaccharides over a reversion of 9 days in a drug-free medium, using HPTLC and MALDI-TOF techniques. Using HPTLC analysis, a constant decrease in the amount of intracellular benzylated oligosaccharides was observed. They were nearly undetectable after four days of culture, in the drug-free medium. However, the decrease was more rapid for slow migrating compounds, indicating a preliminary loss of the sialic acid and fucose residues. This observation was confirmed using the MALDI-TOF technique after methyl esterification of the carboxyl groups of sialic acids (Figure 6). Indeed, after 3 days of reversion, the high mass compounds showed a reduction with a concomitant increase of low mass constituents. The major compound showed a mass with a sodium adduct of 861, corresponding to the benzylated tetrasaccharide Galß14GlcNAcß16[Galß13]GalNAc
-O-bn, whereas its di-sialylated homologue (1472) represented the major MALDI-TOF peak before the reversion. The amount of material dramatically decreased thereafter and a few compounds remained after 8 days of reversion. These minor compounds were somewhat related to the previous compounds since the peak of mass 413 corresponded to Galß13GalN
-O-bn (the N-acetyl group of GalNAc being deleted). The peak at 685 probably corresponded to a hexuronic acid residue attached to the Galß13GalNAc
-O-bn core, a very minor compound actually detected as trace in the accumulated products of HT-29 G cells treated with GalNAc
-O-bn.
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Discussion |
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We previously reported that exposure of the mucin-secreting HT-29 MTX cells to GalNAc-O-bn either as a short-term (24 h) treatment at late confluence, or as a permanent treatment, from 2 days after seeding up to late confluence significantly changed the glycosylation of the mucins (Huet et al., 1995
; Delannoy et al., 1996
; Hennebicq-Reig et al., 1998
). The elongation of the mucin oligosaccharide chains and particularly the addition of sialic acid in the
2,3 linkage to Gal residues was markedly reduced and mucins strongly expressed the Tn and T glycan epitopes. At the same time, both basal and secretagogue-stimulated secretion of the mucins was dramatically inhibited. These observations suggested a possible relation between the glycosylation of mucins and their intracellular trafficking and/or secretion. However, due to the multiple effects of GalNAc
-O-bn in HT-29 cells, several other possible mechanisms could also account for the observed blockade in mucin secretion, and this led us to examine possible modifications of the glycolipid metabolism in HT-29 cells. Our results particularly showed a marked accumulation of several GalNAc
-O-bn derived oligosaccharides in the HT-29 cells permanently treated by GalNAc
-O-bn.
GalNAc-O-bn was previously shown to constitute a very good substrate for the core-1 ß1,3-galactosyltransferase in different types of cultured cells, including HT-29 cells (Kuan et al., 1989
; Huang et al., 1992
; Delannoy et al., 1996
). This point is confirmed in the present study, since all the accumulated compounds (except one) possess the Galß13GalNAc
1- motive (Figure 7). Moreover, the structures of different GalNAc
-O-bn derived oligosaccharides suggest that the formed Galß13GalNAc
-O-bn behaves as a substrate for several other glycosyltransferases: Galß13GalNAc
2,3-sialyltransferase (ST3Gal I), core-2 ß1,6-N-acetyl-glucosaminyltransferase, and a ß1,3-galactosyltransferase which is able to transfer a ß1,3-Gal at the non-reducing end of Galß13GalNAc
-O-bn (Figure 7). The oligosaccharide structure Neu5Ac
23Galß13GalNAc
-O-bn (compound 5) corresponds to the main oligosaccharide structure found on the HT-29 MTX mucins (Hennebicq-Reig et al., 1998
). Furthermore, some of these firstly processed compounds are further elongated. For instance, Neu5Ac
23Galß13GalNAc
-O-bn can be substituted by the Neu5Ac
23Galß13GalNAc (sialyl to GalNAc)
2,6-sialyltransferase (ST6GalNAc III; Sjoberg et al., 1996
), leading to the synthesis of the disialylated form of T-antigen (compound 9-a). GlcNAcß16Galß13GalNAc
-O-bn (compound 4) can be further elongated onto the GlcNAcß1,6-branch, by the sequential addition of a galactose residue (compound 6), and of a sialic acid residue (compound 8) transferred by a ß1,4-galactosyltransferase and the Galß14GlcNAc
2,3-sialyltransferase (ST3Gal IV), respectively. Furthermore, compound 8 is the precursor of compounds 9-b (the sialyl-Lewisx epitope) and 10. These two compounds reflect the catalytic action of either a Neu5Ac
23Galß14GlcNAc
1,3-fucosyltransferase, probably Fuc-TVII which specifically synthesizes the sialyl-Lewisx epitope (Sasaki et al., 1994
; Natsuka et al., 1994
), or ST3Gal I, respectively. Finally, the more complex GalNAc
-O-bn derivative described here (compound 11), which contains both a sialyl-Lewisx epitope on the ß1,6-branch and a sialic acid residue
2,3-linked to the terminal Gal of core 1, reflects the action of both ST3Gal I and Fuc-TVII. Consequently, complex fucosylated oligosaccharide derivatives are synthesized despite the fact that no fucosylated oligosaccharide structure was detected in the mucins purified from HT-29 MTX cells (Hennebicq-Reig et al., 1998
). Indeed,
1,3/1,4 fucosyltransferase activity has been previously detected in the parental HT-29 cell line (Majuri et al., 1995
). Furthermore, among all the identified compounds only the oligosaccharide structures of the compounds 5, 8, 9a, and 10 were previously found in the HT-29 MTX mucins.
The characterization of the Neu5Ac residue 2,3-linked to N-acetyl-lactosamine gives us a new insight into the effect of GalNAc
-O-bn on the sialylation of HT-29 cell glycoproteins. In chronic treatment, we previously observed that GalNAc
-O-bn not only affects the sialylation of O-glycosylated glycoproteins by competing with the action of ST3Gal I, but also the sialylation of other glycoproteins, known mainly as N-glycosylated such as dipeptidylpeptidase IV and carcinoembryonic antigen (Huet et al., 1998
). The accumulation of sialylated, N-acetyl-lactosamine derivatives (compound 8 and derivatives) testifies that GalNAc
-O-bn treatment could also influence the sialylation of N-glycoproteins, by a competition with specific sialyltransferases involved in their elongation. This observation suggests that the inhibition of N-glycosylation processes could be responsible for the alteration of the apical targeting of brush border glycoproteins in differentiated HT-29 cells. Indeed, abnormal N-glycosylation of the growth hormone has been previously shown to turn the secretion on to the apical side in MDCK cells (Scheiffele et al., 1995
).
In parallel with the main biosynthetic pathway of GalNAc-O-bn derivatives, several other minor compounds were observed. Compound 6 may be used as an acceptor substrate by an
3-fucosyltransferase, different from Fuc-TVII leading to compound 7-a, which contains a Lewisx epitope onto the ß1,6-branch. Several
3-fucosyltransferases such as Fuc-TIV, Fuc-TV, Fuc-TVI (Mollicone et al., 1992
), or the newly cloned Fuc-TIX (Kudo et al., 1998
) could be responsible for the biosynthesis of this compound. Compounds 1 and 6 apparently serve as acceptor substrates for a putative ß1,3-galactosyltransferase, to generate the compounds 2 and 7-b, respectively. However the Galß13Galß1- terminal sequence has never been observed in oligosaccharides from mammalian mucins and no UDP-Gal: Galß13GalNAc ß1,3galactosyltransferase has been detected in mammalian tissues or cells till now (Brockhausen, 1995
). But it can be hypothesized that compound 2 is formed by the action of the ß1,3-galactosyltransferase acting on GAGs. An indication that such enzymes can also act on GalNAc
-O-bn derivatives was obtained for the minor compound, containing a hexuronic acid. Further work is necessary to gain more knowledge on this atypical structure.
Surprisingly, even if most compounds described here derive from Galß13GalNAc-O-bn, we could identify the structure of Galß14GlcNAcß16GalNAc
1-O-bn (compound 3) at a significant level. This compound could correspond to a degradation of compound 6 by the action of a specific galactosidase, all the more than the disaccharide GlcNAcß16GalNAc
-O-bn could not be detected (Figure 7). However, core-6 O-glycans occur in human (Hounsell et al., 1985
; Schachter and Brockhausen, 1992
), suggesting the existence of a human-specific core-6 ß1,6-N-acetylglucosaminyltransferase. Such an activity has been described in human ovarian tissue (Yazawa et al., 1986
), although the presence of this enzyme in the human colon has not yet been demonstrated (Brockhausen, 1995
). Since compound 3 is an onco-fetal antigen (Hounsell et al., 1985
; Schachter and Brockhausen, 1992
), it would be of interest to study further such transferase activity in relationship with malignant transformation.
All these compounds were accumulated intracellularly in HT-29 G cells and not significantly externalized except the disaccharide Galß1-3GalNAc-O-bn, at a low level. Reversion experiments indicated that the lifetime of the GalNAc
-O-bn derived oligosaccharides in treated HT-29 cells was a few days. The degradation of the more peripheral monosaccharides occurred in intact cells by the action of neuraminidase(s) and fucosidase(s) first, and then followed by the successive cleavage of other distal monosaccharides.
Such accumulation of GalNAc-O-bn derivatives was neither found inside the Caco-2 cells analyzed in the same conditions, or in the culture medium after permanent treatment. However, after a short time (24 h) exposure of Caco-2 cells to GalNAc
-O-bn, several types of complex benzylated oligosaccharides could be detected in the culture medium with metabolic labeling and chromatography (Byrd et al., 1995
). We have also previously analyzed the metabolism of GalNAc
-O-bn, after 24 h metabolic labeling and HPLC analysis, in HT-29 cells (Delannoy et al., 1996
) and also in Caco-2 cells (data not shown). We could also characterize benzylated oligosaccharides in both types of cells, the disaccharide Galß13GalNAc
-O-bn being the major type. Altogether, these observations show that HT-29 G cells considerably accumulate several benzylated oligosaccharides in comparison to Caco-2 cells. This phenomenon can be related to the marked effects upon the intracellular trafficking of mucins and/or brush border glycoproteins, specifically observed after permanent GalNAc
-O-bn treatment of differentiated HT-29 cells in comparison to Caco-2 cells (Huet et al., 1998
). The amount of benzylated oligosaccharides in HT-29 G cells is enormous (0.51 mg hexose/mg cell protein), and is probably related to the numerous vesicles accumulated in the HT-29 cells upon permanent GalNAc
-O-bn exposure (Huet et al., 1998
). The question now, is to elucidate why HT-29 cells specifically accumulate such a panel of benzylated oligosaccharides. A first possibility is the existence of a high glycosylation potential in these cells, due to a great amount of glycosyltransferases, or to the presence of specific glycosyltransferases, especially a core-1 ß1,3-galactosyltransferase with a high affinity for GalNAc
-O-bn. Another explanation could rely in a reduced degradation of the synthesized oligosaccharide derivatives under GalNAc
-O-bn exposure, resulting from an inhibition of the lysosomal glycosidases, or from a defect in the targeting towards lysosomes.
In addition to these new insights into the mechanisms involved in the alterations of the intracellular trafficking in GalNAc-O-bn treated HT-29 cells, our results show that the HT-29 cells can now be considered as a cell machinery able to efficiently produce a series of endogenously tagged oligosaccharides. Several of the synthesized compounds can be of primary importance for studying the activity and specificity of different glycosyltransferases. But overall, the HT-29 cell system can synthesize benzylated sialyl-Lewisx, which could have importance in the fields of inflammation and cancer.
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Material and methods |
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Cell cultures
Enterocyte-like HT-29 cells (referred as HT-29G) were selected by glucose deprivation (Lesuffleur et al., 1991). Cells were grown in Dulbeccos modified Eagles minimal essential medium (DMEM; Eurobio, Paris, France), supplemented with 10% inactivated (30 min, 56°C) fetal bovine serum (Boehringer, Mannheim, Germany). GalNAc
-O-bn was solubilized in DMEM and added at the final concentration of 2 mM. All experiments and maintenance of the cells were done in 25 or 75 cm2 T flasks (Corning Glassworks, Corning, NY) at 37°C in a 10% CO2/90% air atmosphere. Cells were seeded at 2x104 cells/cm2. The medium was changed daily in all culture conditions. Control Caco-2 cells were cultured as previously reported (Pinto et al., 1983
) and analyzed between passage 80 and 90. The cell viability was evaluated by Trypan blue exclusion and found to be >93% for control and GalNAc
-O-bn treated cells.
Glycolipid extraction and chromatography
In typical experiments, the lyophilized material of the cell pellets (8495 mg protein) or of the culture media was submitted to a Folch extraction and partition as previously described (Zanetta et al., 1980) with the following modifications. The lyophilized powder was transferred to heavy-walled Pyrex tubes with a Teflon-lined screw cap (13 mm external diameter; 8 ml volume) into which a small magnet was introduced. After addition of 66 µl water and 0.825 ml of distilled methanol, the samples were stirred during 5 min, followed by the addition of 1.67 ml of pure chloroform. After 30 min under agitation at room temperature, the magnet was recovered and the tubes were centrifuged during 30 min at 4000 r.p.m. at room temperature. The supernatants were carefully aspirated with a pipette and transferred in other tubes. The pellets were supplemented with 1.32 ml of chloroform-methanol mixture (C/M) 1:2 (v/v) and then stirred for 30 min at room temperature. After centrifugation, the supernatants were pooled to the corresponding ones of the first extraction, and 1.32 ml of chloroform was added. 1.056 ml of a 0.88% KCl solution in water was added and the closed vials were submitted to gentle agitation. The upper phase was recovered by aspiration with a pipette and transferred in identical tubes. The lower phase was supplemented with 1.056 ml of the theoretical upper phase, the tubes were stirred as above and the upper phase was pooled to the previous ones. The pellet was used for protein determinations.
The samples of the upper phase were transferred quantitatively into 50 ml pear-shaped vessels and after addition of 0.5 ml of toluene, they were evaporated to dryness at room temperature with a rotary evaporator. The dry material was recovered by three successive extractions with 500 µl of C/M 1:1 (v/v) and by pipetting with a flat-tip Pasteur pipette (this allows to eliminate most of the KCl crystals). The tubes were centrifuged for 30 min at 4000 rpm at room temperature in order to eliminate the remaining KCl crystals. The supernatants were recovered in identical tubes and evaporated under a stream of nitrogen. The samples of the lower phase were treated in a similar way.
Aliquots of the samples (1 µl) were loaded as a 5 mm large streak on Silicagel 60 HPTLC glass plates and ascending chromatography was performed until the solvent front arrived to 0.5 cm to the top of the plate (45 min) in the solvent methyl acetate/n-propanol/chloroform/methanol/0.25% aqueous KCl 25:20:20:20:17.5 (Zanetta et al., 1980). The plates were revealed using the orcinol/sulfuric acid method for hexoses and/or resorcinol/hydrochloric acid for sialic acids.
Identification of glycolipids found in the upper phase of the Folch extraction
Partial acid hydrolysis.
The compounds found in the upper phase of the Folch partition (10 µg hexoses) were submitted to a mild acid hydrolysis (1.5 h at 80°C in 50 µl of 2 M acetic acid) to remove sialic acid residues. After cooling, the samples were evaporated at room temperature using a rotary evaporator, dissolved in C/M 1:1 (v/v), then submitted to HPTLC in the solvent system described above. The plates were revealed using the orcinol/sulfuric acid and/or the resorcinol hydrochloric acid methods.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analyses.
Aliquots of the glycolipids found in the upper phase of the partition (10 µg hexose) and the products of partial acid hydrolysis (desialylated) were separately placed in a conical Pyrex tubes with a Teflon-lined screw cap, and then lyophilized. The dry samples were supplemented with 20 µl dimethyl sulfoxide and 20 µl of iodomethane and reacted for 2 h at room temperature in the dark (Powell and Harvey, 1996). After evaporation of the excess of iodomethane under a light stream of nitrogen, samples were lyophilized, then solubilized in 20 µl of methanol. They were co-crystallized onto the target with the tri-hydroxy-acetophenone matrix (Papac et al., 1996
). The mass spectra were acquired in the reflection mode under 6 kV accelerating voltage and positive ion detection.
Preparative thin-layer chromatography.
The constituents of the upper phase of the Folch partition were further separated by preparative thin-layer in the solvent mixture methyl-acetate/chloroform/methanol/1-propanol/0.25% aqueous KCl 25/20/20/20/17.5 v/v/v/v/v (Zanetta et al., 1980). The compounds were spotted as a 12-cm-long streak and as a separated 0.5 cm streak and chromatography was performed in the previous solvent. After drying in a cold air stream, the part of the plate containing the 0.5 cm streak was cut with a diamond knife and revealed using the orcinol/sulfuric acid staining. The areas corresponding to the orcinol-positive spots of the other part of the plate were lightly marked with a pencil. The Silicagel was aspirated with a vacuum pump into pipettes closed with glass wool. The material was eluted first with C/M 1:1 then methanol/water (1:3 v/v). The solvent was evaporated under reduced pressure at room temperature and the dry residue was suspended in a small volume of C:M (1:1 v/v) and centrifuged in order to eliminate traces of Silicagel. The pellet was washed in the same mixture and the pooled supernatants were taken up in C:M (1/1).
Gas chromatography/mass spectrometry analysis of the individual compounds.
The putative constituents (monosaccharides, fatty acid methyl esters and sphingosines) of the individual compounds were analyzed in a single step by GC/MS using the electron impact detection mode and quantified by GC/MS as the heptafluorobutyrate derivatives of the O-methyl glycosides obtained after anhydrous acid methanolysis (Zanetta et al., 1999). For GC/MS analysis, the GLC separation was performed on a Carlo Erba GC 8000 gas chromatograph equipped with a 25 m x 0.25 mm CP-Sil5 CB Low bleed/MS capillary column, 0.25 µm film phase (Chrompack France, Les Ullis, France). The temperature of the Ross injector was 280°C and the samples were analyzed using the following temperature program: 90°C for 3 min then 5°C/min until 260°C, followed by a plateau of 20 min at 260°C. The column was coupled to a Finnigan Automass II mass spectrometer (mass limit 1000) for routine analyses.
Nuclear magnetic resonance (1H-NMR)
Before NMR analysis, samples dissolved in C/M (1/1: v/v) were evaporated under a stream of nitrogen, solubilized in D2O then lyophilized. After redissolution of samples in D2O, 1H-NMR spectroscopy was performed on a 400 MHz Brucker ASX 400 WB spectrometer at 300°K and at 315°K. Chemical shifts were determined in ppm downfield from trimethyl silane (0 ppm).
Methylation analysis.
Aliquots of the compounds for which a residual ambiguity remained after NMR analysis were permethylated using methyl iodide in DMSO/NaOH according to Ciucanu and Kerek (1984) for 2 h at room temperature as modified by Michalski and Strecker (1995)
. The samples were submitted to acid methanolysis then acylation with acetic anhydride (Michalski and Strecker, 1995
). The resulting methyl ether glycoside acetates were analyzed by GC/MS in the same conditions as above.
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Acknowledgments |
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Abbreviations |
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Enzymes: the nomenclature of sialyltransferases is based on Tsuji et al. (1996, Glycobiology, 6(7), vvii), the nomenclature of fucosyltransferases is based on Oriol et al. (1999, Glycobiology, 9, 323334), and the nomenclature of the other enzymes involved in O-glycan biosynthesis is based on Brockhausen (1995), In Glycoproteins, J.Montreuil, J.F.G.Vliegenthart, and H.Schachter (eds.), New Comprehensive Biochemistry, Vol. 29a, Elsevier Science B.V., Amsterdam, pp. 201259): core-1 ß3-Gal-T:UDP-Gal:GalNAc-R ß1,3-galactosyltransferase, EC 2.4.1.122; ß4-Gal-T:UDP-Gal:GlcNAc-R ß1,4-galactosyltransferase, EC 2.4.1.38; core-2 ß6-GlcNAc-T:UDP-GlcNAc:Galß13GalNAc-R (GlcNAc to GalNAc) ß1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; core-6 ß6-GlcNAc-T:UDP-GlcNAc:GalNAc-R ß1,6-N-acetylglucosaminyltransferase, EC 2.4.1.-;
3-Fuc-T:GDP-Fuc:Galß14GlcNAc (Fuc to GlcNAc)
1,3-fucosyltransferase, including Fuc-TIV (myeloid enzyme) FUT4-encoded
1,3-fucosyltransferase, Fuc-TV FUT5-encoded
1,3-fucosyltransferase, Fuc-TVI (plasma enzyme, EC 2.4.1.152) FUT6-encoded
1,3-fucosyltransferase, and Fuc-TIX FUT9-encoded
1,3-fucosyltransferase; Fuc-TVII:GDP-Fuc:Neu5Ac
23Galß14GlcNAc
1,3-fucosyltransferase, EC 2.4.1.-; ST3Gal I:CMP-Neu5Ac:Galß13GalNAc
2,3-sialyltransferase, EC 2.4.99.4; ST3Gal IV:CMP-Neu5Ac:Galß14GlcNAc
2,3-sialyltransferase, EC 2.4.99.-; ST6GalNAc I:CMP-Neu5Ac:R-GalNAc
1-O-Ser/Thr
2,6-sialyltransferase, EC 2.4.99.3; ST6GalNAc II:CMP-Neu5Ac:(Neu5Ac
23)01Galß13GalNAc
1-O-Ser/Thr
2,6-sialyltransferase, EC 2.4.99.-; ST6GalNAc III:CMP-Neu5Ac:Neu5Ac
23Galß13GalNAc
2,6-sialyltransferase, EC 2.4.99.7.
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Footnotes |
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References |
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