Glycobiology, 2000, Vol. 10, No. 3 263-271
© 2000 Oxford University Press
Identification of a novel UDP-Glc:GlcNAc ß1
4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains
Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081BT Amsterdam, the Netherlands and 2Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA
Received on June 28, 1999; revised on August 12, 1999; accepted on August 30, 1999.
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Abstract |
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Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L.stagnalis were incubated in media containing either [3H]-mannose, [3H]-glucosamine, or [3H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L.stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L.stagnalis contains a ß1
4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is ß6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is ß2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel ß14-glucosyltransferase present in the prostate gland may be involved in the biosynthesis of Glcß14GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans. Key words: glycoprotein biosynthesis/glycosyltransferase/N-glycan/glycosylation
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Introduction |
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Most of the complex-type N-linked oligosaccharides in mammalian glycoproteins are based on Galß1
4GlcNAc (lacNAc) units, that serve as backbone structural elements. In invertebrates, occurrence of lacNAc units is only occasionally reported, i.e., in Schistosoma mansoni (Srivatsan et al., 1992b
; Van Dam et al., 1994). Instead, a variant of the lacNAc unit in which the Gal residue is replaced by a GalNAc, resulting in a GalNAcß14GlcNAc (lacdiNAc) unit, is found in an increasing number of invertebrates (reviewed in Van den Eijnden et al., 1995, 1997). The characterization of an UDP-GlcNAc:GlcNAc ß4-N-acetylglucosaminyltransferase from the prostate gland of the snail Lymnaea stagnalis suggests that in this snail a third backbone synthesizing enzyme is present that may be involved in the biosynthesis of chitobiose units in complex-type glycans (Bakker et al., 1994, 1997). The backbone structural element is part of the terminal oligosaccharide that often defines the function of the glycan. Regulation of the biosynthesis of these backbone structural elements, as well as the terminating steps, are therefore expected to be critically important for modulation of the final oligosaccharide structures on individual glycoproteins. We therefore are interested in the variations in the backbone structure occurring in different species, and their regulation of expression.
In this study we examined the newly synthesized glycans in the prostate gland of Lymnaea stagnalis and present data that suggest that Glc is present in diantennary N-glycans. This is remarkable in that Glc has not been reported in complex-type N-glycans previously. These results led us to assay the prostate gland for a potential glucosyltransferase activity involved in the biosynthesis of such structures. This resulted in the detection and characterisation of a novel enzyme, an UDP-Glc:GlcNAcß-R ß14-glucosyltransferase (ß4-GlcT), that may be involved in the synthesis of a fourth variant backbone structural unit in complex-type glycans.
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SDS-PAGE and fluorography of radiolabeled prostate glands of Lymnaea stagnalis
Prostate glands were dissected from L.stagnalis and metabolically radiolabeled in vitro with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to radiolabel glycoproteins synthesized by the glands, and allow analyses of the structures of the glycoprotein glycans. To assess incorporation of radiolabel in the glycoproteins, equal amounts of radiolabeled prostate gland were homogenized and analyzed by SDS-PAGE and fluorography. Many glycoproteins were radiolabeled by the [6-3H]galactose precursor, indicating the presence of galactose and/or glucose (Reitman et al., 1982
; Cummings and Kornfeld, 1984) in the glycoproteins (Figure 1). Also numerous glycoproteins were labeled with [6-3H]glucosamine precursor, that can be incorporated in GlcNAc, GalNAc, and sialic acid residues (Cummings et al., 1983). A much weaker radiolabeling was found using [2-3H]mannose precursor, indicating the relatively limited presence of Man and Fuc residues in the radiolabeled glycoproteins (Kornfeld et al., 1978).
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Con A Sepharose column chromatography of glycopeptides
Glycopeptides were prepared by pronase digestion from metabolically labeled prostate glands, and subjected to Con A Sepharose column chromatography (Figure 2). Unbound glycopeptides were designated as the CAI fraction. Bound material was eluted first with 10 mM
-methylglucoside and subsequently with 100 mM -methylmannoside, and designated as CA2 and CA3, respectively (Figure 2A). It has been shown previously that glycopeptides that do not bind to Con A usually consist of O-linked oligosaccharides, complex-type bisected diantennary, tri- and tetraantennary N-linked glycans (CAI fraction), whereas Con A interacts with relatively high affinity with many of the complex-type diantennary N-linked oligosaccharides (CA2 fraction) and with very high affinity with high mannose- and/or hybrid-type N-linked glycans (CA3 fraction) (Ogata et al., 1975; Krusius et al., 1976; Cummings and Kornfeld, 1982; Merkle and Cummings, 1987). The CA2 fraction was subsequently passed over a Bio Gel P6 column (Figure 2B). This resulted in two peaks, CA2-1 and CA2-2, the latter of which showed the sized of a small diantennary complex N-glycan and was chosen for further analysis. The compositions of both the radiolabeled Con A fractions, as well as the unlabeled CA2-2 fraction, were determined by HPLC (Table I). The results show that the CA2-2 glycopeptides contain, next to mannose, fucose, GlcNAc, and Gal, which are expected to be found in diantennary N-linked oligosaccharides, a relatively high amount of Glc, the presence of which was not expected.
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Enzymatic digestion of the ConA-II fraction, radiolabeled with [3H]-Gal
To investigate the nature of the oligosaccharides that contain Glc, glycopeptides in the CA2-2 fraction, labeled with [3H]-Gal, were treated with different exoglycosidases. No release of [3H]-monosaccharides could be detected by treatment of the CA2-2 fraction with
-amylase, indicating that the [3H]-Glc is not part of glycogen, known to bind with relatively high affinity to Con A as well. In contrast, glycopeptides of fractions CA2-1 and CA3 appeared sensitive to -amylase treatment. Treatment of the CA2-2 glycopeptides with -glucosidase, ß-glucosidase, ß-hexosaminidase, -galactosidase, or ß-galactosidase did not result in release of radiolabeled material, suggesting that the radiolabeled glucose/galactose is not accessible to digestion. To establish that the CA2-2 glycopeptides contain diantennary N-linked glycans, they were defucosylated and part of this material digested with Endo F, known to act specifically on diantennary N-glycans. Equal amounts of radioactivity from both the defucosylated and the defucosylated/Endo F treated material were analyzed by HPAEC. The results (Figure 3) show that the endo F treated material eluted as one peak on HPAEC, whereas the glycopeptides that were not treated with Endo F eluted over a broad range of the column, due to peptide heterogeneity. These results suggest that the Glc containing CA2-2 oligosaccharides are mainly present as diantennary N-linked glycans that are inaccessible to digestion with common glycosidases.
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Glucosyltransferase activity in the L.stagnalis prostate gland
Since the structural data described above suggest the presence of glucose in diantennary N-glycans of the prostate gland of L.stagnalis, we have assayed these glands for a potential glucosyltransferase (GlcT) activity involved in the biosynthesis of such structures. A GlcT activity was detected, using GlcNAcß-S-pNP as the acceptor. Optimal assay conditions were determined by varying the pH (pH 5–8) and the concentration of Triton X-100 and Mn2+. Highest enzyme activity was measured in 100 mM cacodylate at pH 7.6, containing 20 mM Mn2+ and 0.5% Triton X-100. The GlcT activity measured in the prostate gland was relatively low compared to the GlcNAcT and GalNAcT activities using the same acceptor substrate, and appeared to be tissue-specific, since the activity was hardly detectable in the albumen gland (Table II).
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Acceptor substrate specificity of the GlcT
GlcT assays were performed using a number of oligosaccharides and a glycopeptide as potential acceptor substrates (Table III). The results show that some substrates carrying a terminal GlcNAc in ß-configuration can serve as an acceptor for the GlcT, whereas GlcNAc in
-anomeric configuration, or GalNAc, Gal, or Glc in ß-anomeric configuration, were not effective as acceptor. The oligosaccharides 10 and 11, representing partial structures of O-linked glycans, as well as 12 and 13, representing partial diantennary N-glycans, appeared to be good acceptors. Also the glycopeptide ag-GP-F4 served as an acceptor, although it was slightly less effective in comparison with the oligosaccharide substrates. HPAEC analysis revealed that the radioactive monosaccharides released by acid hydrolysis of the glucosylated product obtained with glycopeptide 12, could be identified as glucose (results not shown), indicating that the glycosyltransferase activity involved is a true GlcT. The Glcß14GlcNAc structure that was obtained could not be cleaved by ß-glucosidase, indicating that the enzyme used cannot act on this substrate.
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In contrast, oligosaccharides 8 and 9 were not effective as acceptor substrates. These results show that terminal GlcNAc residues that are ß1
6 linked to Gal/GalNAc or ß12 linked to Man, are effective as substrates, whereas GlcNAc residues that are ß13 or ß14 linked are not effective.
The acceptor specificity of the GlcT differs from that of ß4-GalNAcT, that was shown to act on all substrates with terminal ß-linked GlcNAc, although chitobiose was less preferred (Bakker et al., 1997b). The GlcT resembles the recombinant ß4GlcNAcT except for the preference for diantennary N-glycans (Bakker et al., 1997a).
Product characterization
The product formed by incubation of UDP-Glc with a prostate gland extract as the enzyme source and GlcNAcß-O-pNP as acceptor substrate was analyzed by 1D- and 2D-1H-NMR spectroscopy to determine the glycosidic linkage between the Glc and the GlcNAc residue. The 1D-1H-NMR spectrum of the product is shown in Figure 4. 2D-TOCSY spectra with mixing times of 20–100 ms were recorded of the acceptor substrate GlcNAcß-O-pNP as well as the product, affording complete assignment of the protons of each GlcNAc or Glc residue (see Table IV). Compared to GlcNAcß-O-pNP, the GlcNAc H-3, H-4, and H-5 signals of the product show downfield shifts of 
0.15, 0.26, and 0.14, respectively. These shifts are essentially the same as the shifts of 0.17, 0.25, and 0.13, respectively, observed for GlcNAc H-3, H-4, and H-5 when comparing GlcNAcß-OMe (data not shown) and Glcß1–4GlcNAcß-OMe (Hokke et al., 1993). Together with the coupling constant of the Glc H-1 signal (J1,2 8.3 Hz), these data show that the Glc residue is attached to GlcNAcß-O-pNP in a ß1–4-linkage.
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Discussion |
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In studies aimed at the structural characterization of protein-linked glycans of Lymnaea stagnalis, evidence was obtained that glucose is present in a specific subfraction of complex-type N-linked glycans in the prostate gland (CA2-2), known typically to contain diantennary glycans. Due to the low amount of material obtained, and the inaccessibility of the glycopeptides to degradation with common glycosidases, a full structural characterization of the glycan structure has not been performed. Analysis of the monosaccharide composition of this CA2-2 fraction by HPLC revealed next to Glc, the presence of Man, GlcNAc, Fuc, and Gal, all compounds generally found in diantennary N-glycans. As Glc never has been found in complex-type N-linked glycans previously, we set out to assay glucosyltransferase activity in prostate extracts, and found a glucosyltransferase activity toward ß-linked GlcNAc as an acceptor compound. Product characterization by NMR showed that the enzyme could be identified as a UDP-Glc:GlcNAc ß1
4-glucosyltransferase. The enzyme highly prefers a terminal GlcNAc that is ß6 linked to a Gal or GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is ß2-linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymological data, we propose that the ß4-GlcT present in prostate glands is involved in the biosynthesis of Glcß14GlcNAc units in complex-type glycans, in particular in diantennary N-glycans. It can, however, not be excluded that this enzyme is also involved in the biosynthesis of Glcß14GlcNAc units in O-linked glycans.
Several ß4-glucosyltransferases have been described in literature previously, none of them, however, involved in complex-type glycosylation of proteins. A Bacillus coagulans ß4-glucosyltransferase has been reported that catalyzes the conversion of GlcNAc-PP-undecaprenol into Glcß14GlcNAc-PP-undecaprenol, and thus can use a terminal GlcNAc as an acceptor, like the L.stagnalis enzyme. Other bacterial ß4-glucosyltransferases have been described, for example a Neisseria meningitidis enzyme involved in the biosynthesis of lipo-oligosaccharide (LOS) (Kahler et al., 1996) and a cellulose synthase from Acetobacter xylinum (Aloni et al., 1983). A rat liver ß-glucosyltransferase was recently purified and characterized, and the cDNA encoding the human enzyme has been cloned (Ichikawa et al., 1996; Paul et al., 1996). This enzyme catalyzes the transfer of glucose from UDP-Glc to ceramide, the first glycosylation step of glycosphingolipid biosynthesis.
Until now, the only GlcT that has been found to act on N-linked glycans is an -glucosyltransferase, which is postulated to participate in the quality control mechanism of glycoprotein folding and catalyzes the glucosylation of protein-linked, high mannose-type oligosaccharides (Trombetta et al., 1989; Sousa and Parodi, 1995).
Little is known about the structure of invertebrate glycans. The N-linked glycans present on L.stagnalis hemocyanin have been shown to resemble mammalian glycans in many aspects, but also unusual modifications have been shown to occur (Van Kuik et al., 1986, 1987). Several L.stagnalis glycosyltransferases have been characterized, among them enzymes related to mammalian enzymes (Mulder et al., 1991, 1995a, 1995b, 1996; Bakker et al., 1994). The backbone structure of the N-glycans found on hemocyanin was shown to consist of a LacdiNAc unit (Van Kuik et al., 1986, 1987), similarly as has been found in several other invertebrate glycans (Nyame et al., 1989; Srivatsan et al., 1992a; Kubelka et al., 1993, 1995; Hollander et al., 1993; Reason et al., 1994). In accordance with these data, a high ß4-GalNAcT activity was demonstrated in the connective tissue of L.stagnalis, the site of synthesis of hemocyanin (Mulder et al., 1995b).
Based on the size of the diantennary glycan fraction, it is expected that extension of the GlcNAc2Man3 core will be very short, possibly consisting of a terminal GlcNAc, or a backbone structure, i.e., Xß14GlcNAc.
Since little GalNAc could be detected in the CA2-2 glycopeptide fraction, LacdiNAc units are not expected to occur frequently as backbone on diantennary glycans in the prostate gland. As also low amounts of Gal were found, and GalT activity towards GlcNAc is hardly detectable (unpublished results), the occurrence of LacNAc units in the CA2-2 glycopeptides is unlikely. The ß4-GlcNAcT that was cloned from the prostate gland did not act efficiently on diantennary glycan acceptors, but rather preferred terminal GlcNAc that is ß6-linked to Gal or GalNAc, structures occurring in O-linked glycans (Bakker et al., 1994, 1997). It is therefore unlikely that this enzyme plays a role in the synthesis of diantennary N-linked glycans. The acceptor specificity of the ß4-GlcT characterized in this study indicates that this enzyme is able to synthesize Glcß14GlcNAc units in N-linked diantennary glycans in vitro. It might be possible, therefore, that part of the prostate diantennary glycans carry novel Glcß14GlcNAc backbone structural units, a supposition that is supported by the observation that Glc is part of the diantennary glycopeptide fraction.
Interestingly, comparison of the activities in the prostate gland of glycosyltransferases that are able to synthesize a backbone structure (i.e., Xß14GlcNAcß-) revealed that a ß4-GalNAcT was present as the major backbone-synthase activity, in addition to lower ß4GlcNAcT and ß4GlcT activities. This poses the question why hardly any GalNAc can be detected in the N-linked diantennary glycans. Preliminary acceptor specificity studies of the prostate ß4-GalNAcT showed that the enzyme acts efficiently on diantennary glycans in vitro, similarly as has been shown for several other invertebrate ß4-GalNAcTs (Neeleman et al., 1994; Mulder et al., 1995b; Van Die et al., 1996). This suggests that next to the relative activity and acceptor specificity of the glycosyltransferase, other parameters may play a role in regulation of glycoprotein synthesis. It might be that specific protein-motifs are involved. Alternatively, as the L.stagnalis prostate gland is composed of several different cell-types, the proteins containing the diantennary glycans might be expressed in a specific cell-type only, concomitantly with the ß4-GlcT and/or ß4-GlcNAcT, whereas expression of ß4-GalNAcT is limited to another cell-type.
The results described in this manuscript indicate that the prostate gland of L.stagnalis contains a novel ß4-GlcT, potentially involved in the synthesis of unusual diantennary glycans. The data suggest that the regulation of biosynthesis of complex-type glycans in the L.stagnalis prostate gland is complex. Apparently, despite the presence of several glycosyltransferases that are able to act on biantennary glycans in vitro, only specific structures are formed. This seems to be due partly to a restricted acceptor specificity as found for the ß4-GlcNAcT and ß4-GlcT, but other, yet unknown, factors are supposed to play a role as well. This complex regulation might appear to be similar to the mammalian system, in which several different UDP-Gal:GlcNAc ß4-GalTs with yet not fully known properties, in some cell types in addition to a ß4GalNAcT, are supposed to be involved in the synthesis of backbone structures in mammalian protein-linked complex-type glycans (Schachter and Roseman, 1980; Do et al., 1997; Schwientek et al., 1998; Ujita et al., 1998; Van Die et al., 1999; Van den Nieuwenhof et al., 1999). Detailed analysis of the Lymnaea stagnalis protein-linked glycans, in combination with enzymatic studies and comparison to the mammalian situation, might give more insight in the factors that are relevant for the regulation of glycoprotein synthesis in general.
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Materials and methods |
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Animals and materials
Specimens of L.stagnalis were bred under standard laboratory conditions. Acceptor compounds (see Table III) 1, 2, 3, and 5 and GlcNAcß-OMe were purchased from Sigma Chemical Co. (St. Louis, MO), 8, 9, and 11 from Toronto Research Chemicals (Toronto, Ont), 10 from Dr. A.Veyrières (Université Paris Sud, Orsay, France), 12 from Dr. O.Hindsgaul (University of Alberta, Edmonton, Alberta), and 4, 6, and 7 from Koch-Light Laboratories, England.
Glycopeptide GP-F4 was prepared from asialo-fibrinogen by pronase digestion as described previously (Nemansky and Van den Eijnden, 1993). GP-F4 was enzymatically degalactosylated with jack bean ß-galactosidase (0.2 U/µmol terminal Gal) in 50 mM sodium acetate pH 4, to yield the agalacto (ag) form.
UDP[3H]Glc (19.7 Ci/mmol) was obtained from Amersham, UDP[3H]Gal (50 Ci/mmol), UDP[3H]GlcNAc (30.4 Ci/mmol), and UDP[3H]GalNAc (24 Ci/mmol) were purchased from NEN Life Science Products. The sugar nucleotides were diluted with unlabeled UDP-sugars (Sigma) to give the desired specific radioactivity. [2-3H]Mannose (17.6 Ci/mmol), [6-3H]glucosamine (29 Ci/mmol), and [6-3H]galactose (29.5 Ci/mmol) were purchased from NEN Life Science Products. Concanavalin A-Sepharose (Con A-Sepharose) was obtained from Pharmacia Biotech Inc.
Metabolic radiolabeling of L.stagnalis prostate glands and SDS-PAGE of radiolabeled glycoproteins
Prostates were dissected from 75 snails. After washing, these prostates were incubated in vitro with 1 mCi/ml of either [3H]mannose, [3H]glucosamine, or [3H]galactose in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg streptomycin (all from Life Technologies, Inc.), in a final volume of 2 ml for 4 h at 37°C, to radiolabel their oligosaccharides. From each labeling, one prostate gland was taken, washed twice in PBS, and sonicated in SDS-PAGE buffer. The radiolabeled glycoproteins were then analyzed by SDS-PAGE on a 10% acrylamide slab gel (Laemmli, 1970) under reducing conditions. The gel was fixed in an aqueous solution containing 25% (v/v) isopropanol and 10% (v/v) acetic acid and incubated for 40 min in "Amplify" (Amersham) at room temperature and visualized by fluorography.
Preparation of radiolabeled glycopeptides
From each labeling, 20 radiolabeled prostate glands were washed two times with PBS, homogenized and after lipid extraction resuspended in 10 ml PBS. The residual protein was digested with 100 µg pronase E (Sigma) for 72 h at 37°C. Subsequently, the sample was boiled for 30 min and desalted by passage over a column (0.75 40 cm) of Sephadex G25 in water containing 7% n-propanol. The radiolabeled glycopeptides were recovered in the void fractions and freeze-dried.
Con A column chromatography
Radiolabeled glycopeptides were fractionated on a 3 ml column (0.9 5 cm) of Con A-Sepharose at room temperature, as described previously (Cummings and Kornfeld, 1982). Glycopeptides bound at the column were eluted with 10 mM -methylglucoside followed by elution with 100 mM -methylmannoside. Fractions of 2 ml were collected from the column and aliquots of each fraction were taken to determine the radioactivity by liquid scintillation. Radioactive fractions of unbound (CA1), bound (CA2), and strongly bound (CA3) glycopeptides were pooled and desalted by passage over Sephadex as described above. Chromatography of the sample CA2, radiolabeled with [3H]galactose, on Bio-Gel P6 was performed on a 1.6 200 cm column equilibrated in 50 mM ammonium acetate pH 5.2 and 4 ml fractions were collected.
Analysis of sugar composition in radiolabeled oligosaccharides or glycopeptides
Radiolabeled oligosaccharides or glycopeptides were hydrolyzed by heating in 2 M TFA at 100°C for 4 h whereafter the hydrolysates were freeze dried. Samples were analyzed by high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The system consisted of a Dionex Bio-LC gradient pump, a CarboPac MA-1 column (4 250 mm) and a PAD model-2 detector. For separation of the products, isocratic elution was conducted with 0.2 M NaOH at a flow rate of 1 ml/min. Other conditions on chromatography and detection were as described before (Bakker et al., 1994).
Glycosidase treatments
[3H]Galactose labeled glycopeptides were treated with 10 U of -amylase (Bacillus species, Sigma) in 75 µl of 50 mM phosphate buffer, pH 6.8, for 18 h at 37°C. [3H]Galactose labeled glycopeptides were incubated with either 50 mU -glucosidase in 50 mM citrate buffer at pH 6.0, or 50 mU ß-glucosidase in 50 µl 50 mM citrate buffer pH 5.0 for 20 h at 37°C or with either 60 mU -galactosidase (Sigma) in 100 µl 50 mM phosphate buffer pH 6.0, 50 mU jack bean ß-galactosidase (Sigma) in 100 µl 50 mM citrate buffer pH 3.5 or 30 mU bovine testes ß-galactosidase (Sigma) in 100 µl 50 mM citrate buffer pH 4.5 for 20 h at 25°C. To separate released monosaccharides from the glycopeptide fraction after digestion, the samples were applied on a column of Con A-Sepharose (1 ml) run in TBS. The columns were washed three times with 1 ml TBS, and eluted with 100 mM -methylmannoside. The radioactivity of all fractions was determined by liquid scintillation.
Analysis of Endo F treated [3H]galactose labeled glycopeptides by HPLC
[3H]-Labeled glycopeptides were defucosylated by heating in 0.1 N TFA for 75 min at 95°C, freeze dried and dissolved in water. Half of this sample was incubated with 750 mU endoglycosidase F (Boehringer) in a final volume of 500 µl 50 mM phosphate buffer, containing 25 mM EDTA, pH 7.0, and 0.4% Nonidet NP40, for 20 h at 37°C. Both the Endo F treated and the nontreated samples were passed over a Sep-Pak C-18 cartridge to remove the Nonidet, and over a 1.6 200 cm column of Bio-Gel P4 (200–400 mesh), run in 50 mM ammonium acetate pH 5.2. Both samples were analyzed by HPAEC-PAD, consisting of a Dionex Bio-LC gradient pump, a CarboPac PA100 column (4 250 mm) and a PAD model-2 detector. Chromatographic conditions and detection were as described previously (Van Die et al., 1996).
Preparation of the enzyme and glycosyltransferase assays
Three grams of L.stagnalis prostate glands were homogenized in 30 ml 10 mM sodium cacodylate buffer pH 7.6 containing 0.25 M sucrose, 10 mM MnCl2, and 0.5% Triton X-100 and incubated under stirring for 1 h at 4°C. The homogenate was subsequently centrifuged for 1 h at 20,000 x g, and the supernatant recovered as enzyme source. The standard incubation system contained in a volume of 50 µl 25 nmol of either UDP-[3H]Glc (2.5 Ci/mol), UDP-[3H]GlcNAc (1 Ci/mol), UDP-[3H]GalNAc (1 Ci/mol) or UDP-[3H]Gal (1 Ci/mol), 0.2 µmol ATP (to counteract enzymatic degradation of the nucleotide sugar), 50 nmol of oligosaccharide acceptor, 25 µl of prostate extract and 5 µmol sodium-cacodylate buffer pH 7.6 and 1 µmol MnCl2 (for GlcT assays) or 5 µmol sodium-cacodylate buffer pH 7.2 and 2 µmol MnCl2 (for GalNAcT or GlcNAcT assays). After incubation at 37°C for 60 min the radioactive products from 2 and 10 (Table III) were isolated by chromatography of the mixtures on a column (1 ml) of Dowex 1X8 (Cl–-form) as described previously (Neeleman et al., 1994), for substrates with a hydrophobic aglycon, on Sep-Pak C-18 cartridges (Waters, Milford, MA) as described previously (Palcic et al., 1988) and for the diantennary N-glycans 13 and 14 by Con A chromatography (as described under "glycosidase treatments"). The radioactivity incorporated into the acceptor was assayed by liquid scintillation. Control assays lacking the acceptor substrate were carried out to correct for incorporation into endogenous acceptors.
Product characterization
The glucosylated product obtained with substrate 12 was isolated by Con A-Sepharose chromatography and hydrolyzed by incubation in 2 M TFA at 100°C for 4 h. Subsequently, the hydrolysate was freeze-dried and analyzed by HPAEC as described above.
The product obtained by incubation of UDP-Glc with L.stagnalis prostate extract as enzyme source and GlcNAcß-O-pNP as acceptor was analyzed by 1H-NMR spectroscopy. Prior to NMR spectroscopic analysis, samples were exchanged twice in 99.9% 2H2O with intermediate lyophilization. Finally, samples were dissolved in 500 µl 99.95% 2H2O (Merck). 1H-NMR spectra were recorded at 600 MHz on a Bruker AMX2-600 spectrometer (NSR Center, University of Nijmegen, the Netherlands) at a probe temperature of 27°C. Chemical shifts are expressed in ppm by reference to internal acetone ( 2.225) (Vliegenthart et al., 1983). The spectra were recorded and processed as described previously (Hokke et al., 1998).
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Acknowledgments |
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We thank Wietske E.C.M.Schiphorst for expert technical assistance in part of the work and Drs. A.Veyrières (Université Paris Sud, Orsay, France) and O.Hindsgaul (University of Alberta, Edmonton, Alberta) for gift of oligosaccharides.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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Aloni,Y., Cohen,R., Benziman,M. and Delmer,D. (1983) 4-ß-D-Glucosyltransferase (cellulose synthase) from Acetobacter xylinum. A comparison of regulatory properties with those of the membrane-bound form of the enzyme. J. Biol. Chem., 258, 4419–4423.
Bakker,H., Agterberg,M., Van Tetering,A., Koeleman,C.A.M., Van den Eijnden,D.H. and Van Die,I. (1994) A Lymnaea stagnalis gene, with sequence similarity to that of mammalian ß14-galactosyltransferases, encodes a novel UDP- GlcNAc:GlcNAc ß-R ß14-N-acetylglucosaminyltransferase. J. Biol. Chem., 269, 30326–30333.
Bakker,H., Schoenmakers,P.S., Joziasse,D.H., Van Die,I. and Van den Eijnden,D.H. (1997) The substrate specificity of Lymnaea stagnalis UDP-GlcNAc:GlcNAcß-R ß4-N-acetylglucosaminyltransferase indicates its potential function in the synthesis of novel glycan structures in the snail. Glycobiology, 7, 539–548.
Cummings,R.D. and Kornfeld,S. (1982) Fractionation of asparagine-linked oligosaccharides by serial lectin-agarose affinity chromatography: a rapid, sensitive and specific technique. J. Biol. Che7m., 257, 11235–11240.
Cummings,R.D. and Kornfeld,S. (1984) The distribution of repeating [Galß1,4GlcNAcß1,3] sequences in asparigine-linked oligosaccharides of mouse lymphoma cell lines BW5147 and PHAr2.1:Binding of oligosaccharides containing these sequences to immobilized Datura stramonium agglutinin. J. Biol. Chem., 259, 6253–6260.
Cummings,R.D., Kornfeld,S., Schneider,W.J., Hobgood,K.K., Tolleshaug,H., Brown,M.S. and Goldstein,J.L. (1983) Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J. Biol. Chem., 258, 15261–15273.
Do,K.Y., Do,S.I. and Cummings,R.D. (1997) Differential expression of LacdiNac sequences (GalNAcß-14GlcNac-R) in glycoproteins synthesized by chinese hamster ovary and human 293 cells. Glycobiology, 7, 183–194.
Hokke,C.H., Van der Ven,J.G.M., Kamerling,J.P. and Vliegenthart,J.F.G. (1993) Action of rat liver Galß1–4GlcNAc 26–sialyltransferase on Manß1–4GlcNAcß-OMe, GalNAcß1–4GlcNAcß-OMe, Glcß1–4GlcNAcß–OMe and GlcNAcß1–4GlcNAcß–OMe as synthetic substrates. Glycoconjugate J., 10, 82–90.
Hokke,C.H., Neeleman,A.P., Koeleman,C.A.M. and Van den Eijnden,D.H. (1998) Identification of an 3–fucosyltransferase and a novel 2-fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata. Glycobiology, 8, 393–406
Hollander,T., Aeed,P.A. and Elhammer,A.P. (1993) Characterization of the oligosaccharide structures on bee venom phospholipase A2. Carbohydr. Res., 247, 291–297.[ISI][Medline]
Ichikawa,S., Sakiyama,H., Suzuki,G. and Hidari,K.I.P.J. (1996) Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis. Proc. Natl Acad. Sci. USA, 93, 4638–4643.
Kahler,C.M., Carlson,R.W., Rahman,M.M., Martin,L.E. and Stephens,D.S. (1996) Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis. J. Bacteriol., 178, 6677–6684.
Kornfeld,S., Li,E. and Tabas,I. (1978) The synthesis of complex-type oligosaccharides. II Characterization of the processing intermediates in the synthesis of the complex oligosaccharide units of the vesicular stomatitus virus G protein. J. Biol. Chem., 253, 7771–7778.
Krusius,T., Finne,J. and Rauvala,H. (1976) The structural basis of the different affinities of two types of acidic N-glycosidic glycopeptides for concanavalin A-sepharose. FEBS Lett., 72, 117–120.
Kubelka,V., Altmann,F., Staudacher,E., Tretter,V., März,L., Hård,K., Kamerling,J.P. and Vliegenthart,J.F.G. (1993) Primary structures of the N-Linked carbohydrate chains from honeybee venom phospholipase-A2. Eur. J. Biochem., 213, 1193–1204.[ISI][Medline]
Kubelka,V., Altmann,F. and März,L. (1995) The asparagine-linked carbohydrate of honeybee venom hyaluronidase. Glycoconjugate J., 12, 77–83.[ISI][Medline]
Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[Medline]
Merkle,R.K. and Cummings,R.D. (1987) Lectin affinity chromatography of glycopeptides. Methods Enzymol., 138, 232–259.[ISI][Medline]
Mulder,H., Schachter,H., De Jong-Brink,M., Van der Ven,J.G.M., Kamerling,J.P. and Vliegenthart,J.F.G. (1991) Identification of a novel UDP-Gal:GalNacß1–4GlcNac-R ß1–3- galactosyltransferase in the connective tissue of the snail Lymnaea stagnalis. Eur. J. Biochem., 201, 459–465.[ISI][Medline]
Mulder,H., Dideberg,F., Schachter,H., Spronk,B.A., De Jong-Brink,M., Kamerling,J.P. and Vliegenthart,J.F.G. (1995a) In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis the incorporation of GlcNAc by ß2-GlcNAc-transferase I is an essential prerequisite for the action of ß2-GlcNAc-transferase II and ß2-Xyl-transferase. Eur. J. Biochem., 232, 272–283.[ISI][Medline]
Mulder,H., Spronk,B.A., Schachter,H., Neeleman,A.P., Van den Eijnden,D.H., De Jong-Brink,M., Kamerling,J.P. and Vliegenthart,J.F.G. (1995b) Identification of a novel UDP-GalNAc:GlcNAcß-R ß1–4 N- acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis. Eur. J. Biochem., 227, 175–185.
Mulder,H., Schachter,H., Thomas,J.R., Halkes,K.M., Kamerling,J.P. and Vliegenthart,F.G. (1996) Identification of a GDP-Fuc-Gal-ß13GalNAc-R (Fuc to Gal) 12-fucosyltransferase and a GDP-Fuc-Gal-ß14GlcNAc (Fuc to Glcnac) 13-fucosyltransferase in connective tissue of the snail Lymnaea stagnalis. Glycoconjugate J., 13, 107–113.[ISI][Medline]
Neeleman,A.P., Van der Knaap,W.P.W. and Van den Eijnden,D.H. (1994) Identification and characterization of a UDP-GalNAc:GlcNAcß-R ß14-N-acetylgalactosaminyltransferase from cercariae of the schistosome Trichobilharzia ocellata. Catalysis of a key step in the synthesis of N,N"- diacetyllactosediamino (lacdiNAc)-type glycans. Glycobiology, 4, 641–651.
Nemansky,M. and Van den Eijnden,D.H. (1993) Enzymatic characterization of CMP-NeuAc-Gal-ß1–4GlcNAc- R (2–3)-sialyltransferase from human placenta. Glycoconjugate J., 10, 99–108.
Nyame,K., Smith,D.F., Damian,R.T. and Cummings,R.D. (1989) Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal ß-linked N-acetylgalactosamine. J. Biol. Chem., 264, 3235–3243.
Ogata,S., Muramatsu,T. and Kobata,A. (1975) Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose. J. Biochem (Tokyo), 78, 687–696.
Palcic,M.M., Heerze,L.D., Pierce,M. and Hindsgaul,O. (1988) The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays. Glycoconjugate J., 5, 49–63.
Paul,P., Kamisaka,Y., Marks,D.L. and Pagano,R.E. (1996) Purification and characterization of UDP-glucose:ceramide glucosyltransferase from rat liver golgi membranes. J. Biol. Chem., 271, 2287–2293.
Reason,A.J., Ellis,L.A., Appleton,J.A., Wisnewski,N., Grieve,R.B., McNeil,M., Wassom,D.L., Morris,H.R. and Dell,A. (1994) Novel tyvelose-containing tri- and tetra-antennary N-glycans in the immunodominant antigens of the intracellular parasite Trichinella spiralis. Glycobiology, 4, 593–603.
Reitman,M.L., Trowbridge,I.S. and Kornfeld,S. (1982) A lectin-resistent mouse lymphoma cell line is deficient in glucosidase II, a glycoprotein-processing enzyme. J. Biol. Chem., 257, 10357–10363.
Schachter,H. and Roseman,S. (1980) Mammalian glycosyltransferases. Their role in the synthesis and function of complex carbohydrates and glycolipids. In Lennarz,W.J. (ed.), The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, New York, pp. 85–160.
Schwientek,T., Almeida,R., Levery,S.B., Holmes,E.H., Bennett,E. and Clausen,H. (1998) Cloning of a novel member of the UDP-galactose:ß-N-acetylglucosamine ß1,4-galactosyltransferase family, ß4Gal-T4, involved in glycosphingolipid biosynthesis. J. Biol. Chem., 273, 29331–29340.
Sousa,M. and Parodi,A.J. (1995) The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. EMBO J., 14, 4196–4203.[ISI][Medline]
Srivatsan,J., Smith,D.F. and Cummings,R.D. (1992a) Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal ß-linked N-acetylgalactosamine. Glycobiology, 2, 445–452.
Srivatsan,J., Smith,D.F. and Cummings,R.D. (1992b) The human blood fluke Schistosoma mansoni synthesizes glycoproteins containing the Lewis x antigen. J. Biol. Chem., 267, 20196–20203.
Trombetta,S.E., Bosch,M. and Parodi,A.J. (1989) Glucosylation of glycoproteins by mammalian, plant, fungal and trypanosomal protozoa microsomal membranes. Biochemistry, 28, 8108–8116.[Medline]
Ujita,M., McAuliffe,J., Schwientek,T., Almeida,R., Hindsgaul,O., Clausen,H. and Fukuda,M. (1998) Synthesis of poly-N-acetyllactosamine in core 2 branched O-glycans. J. Biol. Chem., 273, 34843–34849.
Van Dam,G.J.,Bergwerff,A.A., Thomas-Oates,J.E., Rotmans,J.P., Kamerling,J.P., Vliegenthart,J.F.G. and Deelder,A.M. (1994) The immunologically reactive O-linked polysaccharide chains derived from circulating cathodic antigen isolated from the human blood fluke Schistosoma mansoni have Lewis x as repeating unit. Eur. J. Biochem., 225, 467–482[ISI][Medline]
Van den Eijnden,D.H., Neeleman,A.P., Van der Knaap,W., Bakker,H., Agterberg,M. and Van Die,I. (1995) Novel glycosylation routes for glycoproteins: the lacdiNAc pathway. Biochem. Soc. Trans., 23, 175–179.[ISI][Medline]
Van den Eijnden,D.H., Bakker,H., Neeleman,A.P., Van den Nieuwenhof,I.M. and Van Die,I. (1997) Novel pathways in complex-type oligosaccharide synthesis—new vistas opened by studies in invertebrates. Biochem. Soc. Trans., 25, 887–893.[ISI][Medline]
Van den Nieuwenhof,I.M., Schiphorst,W.E.C.M., Van Die,I. and Van den Eijnden,D.H. (1999) Bovine mammary gland UDP-GalNAcß-R ß14-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with -lactalbumin. Glycobiology, 9, 115–123
Van Die,I., Van Tetering,A., Bakker,H., Van den Eijnden,D.H. and Joziasse,D.H. (1996) Glycosylation in Lepidopteran insect cells: identification of a ß14-N-acetylgalactosaminyltransferase involved in the synthesis of complex-type oligosaccharide chains. Glycobiology, 6, 157–164.
Van Die,I., Van Tetering,A., Schiphorst,W.E.C.M., Sato,T., Furukawa,K. and Van den Eijnden,D.H. (1999) The acceptor substrate specificity of human ß4-galactosyltransferase V indicates its potential function in O-glycosylation. FEBS Lett., 450, 52–56.[ISI][Medline]
Van Kuik,J.A., Sijbesma,R.P., Kamerling,J.P., Vliegenthart,J.F.G. and Wood,E.J. (1986) Primary structure of a low-molecular-mass N-linked oligosaccharide from hemocyanin of Lymnaea stagnalis. 3-O-methyl-D-mannose as a constituent of the xylose-containing core structure in an animal glycoprotein. Eur. J. Biochem., 160, 621–625.[ISI][Medline]
Van Kuik,A., Sijbesma,R.P., Kamerling,J.P., Vliegenthart,J.F.G. and Wood,E.J. (1987) Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis. ß-O-Methyl-D-galactose and N-acetyl-D-galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein. Eur. J. Biochem., 169, 399–411.[ISI][Medline]
Vliegenthart,J.F.G., Dorland,L. and Van Halbeek,H. (1983) High-resolution, 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem., 41, 209–374.[ISI]
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