Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands
Received on December 19, 2003; revised on January 22, 2004; accepted on January 27, 2004
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Abstract |
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Key words: donor specificity / Sda determinant / Tamm-Horsfall glycoprotein
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Introduction |
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THp has a very heterogeneous glycosylation pattern, distributed over seven N-glycosylation sites (van Rooijen et al., 1999) and one or more O-glycosylation sites (Easton et al., 2000
). Detailed structural studies of the N-glycosylation pattern of THp from individual donors by 1H-nuclear magnetic resonance (NMR) spectroscopy have resulted in the elucidation of 63 complex-type (Hård et al., 1992
; van Rooijen et al., 1998a
,b
) and 4 oligomannose-type (Dall'Olio et al., 1988
; Smagula et al., 1990
; van Rooijen et al., 1999
) N-glycans. Di-, tri-, and tetraantennary structures (including dimeric N-acetyllactosamine sequences) are present, which can be fucosylated, sialylated, and/or sulfated. Structural studies of the O-glycosylation pattern of THp from individual donors by mass spectrometry (MS) have shown the presence of sialylated or fucosylated core 1-type O-glycans (Easton et al., 2000
).
One of the interesting terminal epitopes present in THp N-glycans is the Sda determinant (Donald et al., 1983; Hård et al., 1992
). The Sda pentasaccharide Neu5Ac(
2-3) [GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal can be released by endo-ß-galactosidase digestion of the glycoprotein, together with the tetrasaccharide Neu5Ac(
2-3)Gal(ß1-4) GlcNAc(ß1-3)Gal and the trisaccharide Gal(ß1-4)GlcNAc (ß1-3)Gal (Donald et al., 1983
). In an earlier investigation on THp probes from four male donors, we demonstrated that the molar ratio of Sda pentasaccharide to tetrasaccharide and thus the Sda-related glycosylation is a donor-specific feature (van Rooijen et al., 1998a
). The donor specificity holds also for the total content of Sda pentasaccharide plus tetrasaccharide (van Rooijen et al., 1998a
). Having found this, the question arose whether in genetically identical individuals an identical donor specificity will be observed.
In the present study, THp was isolated from the urine of each individual of a female (pair A) and a male (pair B) monozygotic pairs of twins, and the endo-ß-galactosidase released oligosaccharides were analyzed for their Sda pentasaccharide/tetrasaccharide contents and their molar ratios.
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Results |
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In Figure 5 the HPAEC-PAD fractionation patterns of Superdex-75 fraction II, containing the Sda pentasaccharide II.a and the tetrasaccharide II.b (van Rooijen et al., 1998a), related to the female pair of twins (Figure 5A) and the male pair of twins (Figure 5B), are presented. Based on integrated peak areas of three injections relative to an internal standard the following percentages were found for the Sda pentasaccharide: pair of twins A, 26.8% ± 1.5% (A1) and 24.9% ± 1.5% (A2); pair of twins B, 39.6% ± 1.5% (B1) and 41.2% ± 1.5% (B2). These data also clearly indicate the equality of THp A1 and A2, and of THp B1 and B2, when focused on the molar ratio of the Sda pentasaccharide and the precursor tetrasaccharide.
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Discussion |
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In an earlier study we reported on the donor specificity in relation to the Sda epitope expression for THp in four nongenetically related male donors, yielding HPAEC-PAD molar ratios for II.a:II.b of 50:50, 35:65, 41:59, and 28:72, respectively (van Rooijen et al., 1998a). In the present study the Sda pentasaccharide (II.a):tetrasaccharide (II.b) molar ratios in THp samples from a female (pair A) and a male (pair B) monozygotic pair of twins were estimated, being 38:62 (HPLC-FD)/26:74 (HPAEC-PAD) for pair A and 47:53 (HPLC-FD)/40:60 (HPAEC-PAD) for pair B. As discussed, the ratios obtained via the HPLC-FD approach are considered more reliable. Overall it can be concluded that the ratio between the two motifs is conserved for monozygotic pairs of twins.
The final step in the biosynthesis of the Sda determinant consists of the transfer of a GalNAc residue from UDP-GalNAc to O-4 of the Gal unit in a Neu5Ac(2-3)Gal(ß1-4)GlcNAc(ß1- sequence and is catalyzed by a specific ß-1,4-N-Acetylgalactosaminyltransferase. The substrate specificity of the transferase is very high, that is, the presence of a sialic acid residue at O-3 of the Gal unit is a prerequisite for N-acetylgalactosaminylation (Serafini-Cessi and Dall'Olio, 1983
; Serafini-Cessi et al., 1986
). ß-1,4-N-Acetylgalactosaminyltransferase activity has been demonstrated in microsomal preparations of human and guinea pig kidneys (Piller et al., 1986
; Serafini-Cessi et al., 1986
), whereas a soluble form was found in human colon carcinoma cells (Serafini-Cessi et al., 1995
). Conservation of the molar ratio of the Sda pentasaccharide and the substrate tetrasaccharide in monozygotic pairs of twins, as found in the present study, reveals that the regulation of the ß-1,4-N-acetylgalactosaminyltransferase activity results in a very closely related pattern of glycosylation in genetically homogeneous individuals when it comes to the conversion of the Neu5Ac(
2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1- into the Neu5Ac(
2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1- sequence.
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Materials and methods |
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Endo-ß-galactosidase digestion and oligosaccharide isolation
To 50 mg lyophilized THp, dissolved in 50 mL 30 mM sodium acetate buffer pH 5.9, were added 25 mU endo-ß-galactosidase (from Bacteroides fragilis). The mixture was incubated for 48 h at 37°C then fractionated on a Superdex-75 column (1.6 x 60 cm, Pharmacia) using a FPLC LCC-500 system (Pharmacia, Uppsala, Sweden). The elution was carried out with 50 mM NH4HCO3 at a flow rate of 1 mL/min and monitored by UV absorbance at 214 nm (Pharmacia UV-1/214). UV-positive fractions were lyophilized and checked for the presence of carbohydrate by MALDI-TOF MS and 500-MHz 1D 1H-NMR spectroscopy. MALDI-TOF MS measurements were carried out in the negative-ion mode (matrix: 10 mg/mL 2,4,6-trihydroxyacetophenone in water:acetonitrile = 1:1) (Papac et al., 1996) on a Voyager-DE PerSeptive Biosystems instrument operating at an accelerating voltage of 20 kV (grid voltage 90%, ion guide wire voltage 0.03%) and equipped with a VSL-337ND-N2 laser. 1H-NMR measurements were carried out on a Bruker DRX-500 instrument in D2O at 300 K with suppression of the residual water signal by applying a water-eliminated Fourier transform pulse sequence (Hård et al., 1992
).
Prior to further analysis, the carbohydrate-containing fraction II was redissolved in 0.5 mL water and divided into five 100-µL aliquots.
Determination of the total amount of sialyloligosaccharides in fraction II for each donor was carried out by gas-liquid chromatography. The amounts calculated for the THp A1/A2 and B1/B2 samples are 865/670 and 771/886 µg oligosaccharides/50 mg glycoprotein, respectively, showing only minor differences for the members of each pair of twins, being different from values obtained for other donors (van Rooijen et al., 1998a).
Fluorescent labeling of oligosaccharides with 2-AB and analysis by HPLC
Oligosaccharides were fluorescently labeled with 2-AB as described (Bigge et al., 1995). Briefly, 31.75 mg NaCNBH3 were added to 23.6 mg 2-AB, dissolved in 500 µL dimethyl sulfoxide containing 30% acetic acid. Lyophilized oligosaccharide samples (10 µL of a 100-µL aliquot of a carbohydrate-positive Superdex-75 fraction) in 6 µL water were mixed with 8 µL of this solution and incubated for 2 h at 65°C. For cleaning up the mixture, two disks of QM-A quartz microfibre filters (Whatman) were placed at the bottom of small syringe-shaped glass holders. The filters were washed with 1 mL water, 1 mL 30% acetic acid, and 1 mL acetonitrile; the carbohydrate-containing solutions were loaded and left to dry for 15 min. After washing with 8 mL acetonitrile, the mixtures of labeled oligosaccharides were eluted with 4 x 0.5 mL water, and the combined eluates were lyophilized and redissolved in 40 µL water.
For checking the quantitative conversion of the labeling procedure, MALDI-TOF MS analysis in the negative-ion mode (matrix: 10 mg/mL 2,5-dihydroxybenzoic acid in water) was performed on a small part of each 2-AB-labeled sialyloligosaccharide mixture.
Molar ratios of endo-ß-galactosidase-released, 2-AB-labeled tetra- and Sda pentasaccharides were determined in triplo on GlycoSepC (4.6 x 100 mm) and GlycoSepN (4.6 x 100 mm) columns (Oxford Glycosciences) using a Waters 2690 XE instrument equipped with a Waters 474 fluorescence detector (exc.max = 373 nm,
em.max = 420 nm). For weak anion exchange chromatography on GlycoSepC, elutions were carried out using 20% acetonitrile in water (v/v) (solvent A) and 20% acetonitrile:30% water (v/v):50% 500 mM ammonium formate pH 4.4 (solvent B). Gradient conditions were as follows: t = 0 min, 100% A and 0% B; t = 40 min, 0% A and 100% B; t = 45 min, 0% A and 100% B; t = 46 min, 100% A and 0% B; t = 60 min, 100% A and 0% B. The total run time was 60 min, and the flow rate was 0.4 mL/min. For normal-phase chromatography on GlycoSepN, elutions were carried out using 80% acetonitrile : 20% 50 mM ammonium formate pH 4.4 (v/v) (solvent C), and 50 mM ammonium formate pH 4.4 (solvent D). Gradient conditions were as follows: t = 0 min, 93.5% C and 6.5% D; t = 100 min, 56.2% C and 43.8% D; t = 104 min, 0% C and 100% D; t = 109 min, 0% C and 100% D; t = 111 min, 93.5% C and 6.5% D; t = 140 min, 93.5% C and 6.5% D. The total run time was 140 min, and the flow rate was 0.8 mL/min.
Oligosaccharide analysis by HPAEC
Molar ratios of endo-ß-galactosidase-released tetra- and Sda pentasaccharides were determined by HPAEC-PAD. The Dionex LC instrument consisted of a Dionex Bio LC quaternary gradient module, a PAD 2 detector, and a CarboPac PA-1 pellicular anion-exchange column (25 x 0.9 cm, Dionex, Sunnyvale, CA). For the analysis, 5 µL of a 100-µL aliquot of carbohydrate-positive Superdex-75 fraction was used. Elutions were carried out with a linear concentration gradient of sodium acetate in 0.1 M NaOH as shown in the figures, at a flow rate of 4 mL/min. Galacturonic acid (Sigma, St. Louis, MO) in a final concentration of 0.3 mg/mL was added to the samples as an internal standard (Thurl et al., 1996). Molar ratios were determined by comparison of integrated peak areas from three injections (van Rooijen et al., 1998a
).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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