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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Key words: glycoprotein biosynthesis/glycosyltransferase/N-glycan/glycosylation
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Introduction |
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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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Results |
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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., 1997
a).
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 20100 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 Glcß14GlcNAcß-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 ß14-linkage.
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Discussion |
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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 Glcß1
4GlcNAc units in N-linked diantennary glycans in vitro. It might be possible, therefore, that part of the prostate diantennary glycans carry novel Glcß1
4GlcNAc 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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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 Dulbeccos modified Eagles 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 (200400 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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Footnotes |
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References |
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