Donor specificity in the glycosylation of Tamm-Horsfall glycoprotein: Conservation of the Sda determinant in pairs of twins

Philippe F. Rohfritsch, Meike Rinnbauer, Johannes F.G. Vliegenthart and Johannis P. Kamerling1

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The content of the Sda determinant in urinary human Tamm-Horsfall glycoprotein (THp) has been reported to be donor-specific. This feature was further addressed by investigating THp from genetically identical individuals. To this end, THp was isolated from the urine of two monozygotic pairs of twins (A and B). The four samples (THp A1, A2, B1, and B2) were subjected to endo-ß-galactosidase from Bacteroides fragilis leading to the liberation of the Neu5Ac({alpha}2-3)Gal (ß1-4)GlcNAc(ß1-3)Gal and Neu5Ac({alpha}2-3)[GalNAc(ß1-4)] Gal(ß1-4)GlcNAc(ß1-3)Gal (Sda epitope) motifs, both located at the nonreducing termini of complex type N-glycans. The isolated mixtures of oligosaccharides were analyzed for the absolute and relative amounts of the two oligosaccharides. The obtained data clearly indicate that in THp A1 and A2, and in THp B1 and B2, the molar ratios of the tetra- and Sda pentasaccharide are identical for a pair of twins. This conservation of molar ratios points to an identical relative expression of ß-1,4-N-acetylgalactosaminyltransferase activity involved in the biosynthesis of the Sda determinant. Apparently, the degree of conversion of the tetrasaccharidic Sda precursor into the final pentasaccharidic Sda form can be considered to result from a very closely related pattern of glycosylation for genetically homogeneous individuals.

Key words: donor specificity / Sda determinant / Tamm-Horsfall glycoprotein


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Tamm-Horsfall glycoprotein (THp) is the most abundant protein constituent of normal human urine, being excreted in quantities of up to 100 mg/day (Tamm and Horsfall, 1950Go). It is synthesized in the kidney as a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein (Rindler et al., 1990Go), where it is localized in the thick ascending limbs of the loop of Henle and the early distal convoluted tubule of the nephron. The GPI-anchored form is cleaved by a highly specific protease yielding the urinary form (Cavallone et al., 2001Go; Fukuoka and Kobayashi, 2001Go). Over the years a variety of biological functions for THp have been proposed, for example, natural inhibition of urinary tract bacterial infections (Pak et al., 2001Go), regulation of water/electrolyte transport (Mattey and Naftalin, 1992Go), a role in kidney stone formation (Scurr and Robertson, 1986Go), and immunomodulation (Horton et al., 1990Go; Moonen and Williamson, 1987Go; Muchmore and Decker, 1985Go; Su and Yeh, 1999Go; Thomas et al., 1993Go; Toma et al., 1994Go).

THp has a very heterogeneous glycosylation pattern, distributed over seven N-glycosylation sites (van Rooijen et al., 1999Go) and one or more O-glycosylation sites (Easton et al., 2000Go). 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., 1992Go; van Rooijen et al., 1998aGo,bGo) and 4 oligomannose-type (Dall'Olio et al., 1988Go; Smagula et al., 1990Go; van Rooijen et al., 1999Go) 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., 2000Go).

One of the interesting terminal epitopes present in THp N-glycans is the Sda determinant (Donald et al., 1983Go; Hård et al., 1992Go). The Sda pentasaccharide Neu5Ac({alpha}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({alpha}2-3)Gal(ß1-4) GlcNAc(ß1-3)Gal and the trisaccharide Gal(ß1-4)GlcNAc (ß1-3)Gal (Donald et al., 1983Go). 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., 1998aGo). The donor specificity holds also for the total content of Sda pentasaccharide plus tetrasaccharide (van Rooijen et al., 1998aGo). 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.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Isolation of the oligosaccharide mixture from endo-ß-galactosidase-treated THp
For the isolation of THp, morning urines from two pairs of twins were collected. The four THp samples obtained, A1 and A2 for the female twins pair and B1 and B2 for the male twins pair, appeared as a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) having an apparent molecular mass of 94 kDa (Figure 1A). Monosaccharide analysis (Table I) revealed the presence of Fuc, Man, Gal, GalNAc, GlcNAc, and Neu5Ac in accordance with literature data (Hård et al., 1992Go). The four probes—A1, A2, B1, and B2—were subjected to endo-ß-galactosidase digestion (van Rooijen et al., 1998aGo), and the digestion products were fractionated by size-exclusion chromatography on Superdex-75, yielding similar peak profiles. A typical pattern showing three fractions, denoted I, II, and III, is depicted in Figure 1B. Fraction I contains partially deglycosylated THp glycoprotein, fraction II the released oligosaccharides, and fraction III salts.



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Fig. 1. (A) SDS–PAGE of purified Tamm-Horsfall glycoprotein from two monozygotic pairs of twins; THp A1 and A2 are isolated from individual urines of the female pair, THp B1 and B2 are isolated from individual urines of the male pair. (B) Separation pattern on Superdex-75 after treatment of THp A2 with endo-ß-galactosidase. Fraction II contains the Sda pentasaccharide and precursor tetrasaccharide.

 

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Table I. Monosaccharide analysis data of THp samples isolated from two monozygotic pairs of twins.

 
The presence of the requested oligosaccharide material in fraction II was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS in the negative-ion mode (Figure 2A) and 1H-NMR spectroscopy (Figure 3). MALDI-TOF analysis gave pseudo-molecular ions at m/z 1036.82 ([M–H]) and m/z 834.24 ([M–H]), corresponding with the Sda pentasaccharide Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal (II.a) and the precursor tetrasaccharide Neu5Ac({alpha}2-3) Gal(ß1-4)GlcNAc(ß1-3)Gal (II.b), respectively. The 1H-NMR assignments of the Sda pentasaccharide and the tetrasaccharide (Table II) are based on literature data (van Rooijen et al., 1998aGo). The presence of II.a and II.b is reflected by their typical structural-reporter-group signals (II.a: Neu5Ac H-3e, {delta} 2.671; Neu5Ac H-3a, {delta} 1.923; Gal-c H-3, {delta} 4.156; GalNAc H-1, {delta} 4.75; GalNAc NAc, {delta} 2.013. II.b: Neu5Ac H-3e, {delta} 2.758; Neu5Ac H-3a, {delta} 1.796; Gal-c H-3, {delta} 4.115).



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Fig. 2. MALDI-TOF spectra (negative-ion mode) of the mixture of oligosaccharides, obtained after digestion of THp A2 with endo-ß-galactosidase and subsequent fractionation on Superdex-75 (fraction II). II.a: Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal; II.b: Neu5Ac({alpha}2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal. (A) Free oligosaccharide mixture; (B) 2-AB-labeled oligosaccharide mixture.

 


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Fig. 3. Resolution-enhanced 500-MHz 1D 1H-NMR spectrum of oligosaccharides, obtained after digestion of THp A2 with endo-ß-galactosidase and subsequent fractionation on Superdex-75 (fraction II). II.a: Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal; II.b: Neu5Ac({alpha}2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal.

 

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Table II. 1H-chemical shifts of structural-reporter-group protons of the constituent monosaccharides of the liberated Sda pentasaccharide II.a and the precursor tetrasaccharide II.b (Superdex-75 fraction II) derived from THp

 
Molar ratios of Sda pentasaccharide and tetrasaccharide
The molar ratios of the Sda pentasaccharide Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal (II.a) and the precursor tetrasaccharide Neu5Ac({alpha}2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal (II.b) released from THp of each of the four donors (A1, A2, B1, B2) were estimated along two routes. Mixtures of 2-aminobenzamide (2-AB)-labeled oligosaccharides were analyzed by high-performance liquid chromatography with fluorescence detection (HPLC-FD) on GlycoSepC and GlycoSepN, and mixtures of free oligosaccharides were analyzed by high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on CarboPac PA-1. The results are presented in Table III.


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Table III. Molar ratio determinations of Sda pentasaccharide (II.a) and precursor tetrasaccharide (II.b), released from THp samples A1, A2, B1, and B2 of two monozygotic pairs of twins

 
In Figure 4 the HPLC-FD fractionation patterns of Superdex-75 fraction II after labeling with 2-AB containing the Sda pentasaccharide (II.a) and the tetrasaccharide (II.b), related to the female pair of twins A (GlycoSepC, Figure 4A; GlycoSepN, Figure 4C) and the male pair of twins B (GlycoSepC, Figure 4B; GlycoSepN, Figure 4D), are presented. Following such a protocol, it is important to check the completeness of the derivatization reaction. As shown in Figure 2B, MALDI-TOF analysis in the negative-ion mode gave pseudo-molecular ions at m/z 1157.64 ([M–H]) and m/z 954.65 ([M–H]), corresponding to 2-AB-labeled II.a and II.b, respectively. No indications for non-2-AB-labeled components were found, indicating a complete derivatization.



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Fig. 4. HPLC Fractionation patterns of Superdex-75 fractions II after derivatization with 2-AB on GlycoSepC and GlycoSepN with fluorescence detection. (A) GlycoSepC: THp A1 (gray) and A2 (black) derived samples; (B) GlycoSepC: THp B1 (gray) and B2 (black) derived samples; (C) GlycoSepN: THp A1 (gray) and A2 (black) derived samples; (D) GlycoSepN: THp B1 (gray) and B2 (black) derived samples. For GlycoSepC chromatography, fetuin oligosaccharides were used as standard (light gray) for calibration according to their sialic acid charges. For elution details, see Materials and methods. II.a: Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal; II.b: Neu5Ac({alpha}2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal.

 
As is evident from the GlycoSepC patterns in Figure 4A and 4B, the derivatized oligosaccharides partly overlap, with retention times of 12.1 and 12.5 min for components II.a and II.b, respectively, and are therefore not suited for the determination of the molar ratios. The GlycoSepN patterns show a much better resolution, allowing a quantitative analysis of the molar ratios of II.a and II.b. The retention time of II.b is 21 min (4.2 glucose units [gu]) and that of II.a 25 min (4.7 gu) (Figure 4C and 4D). The assignments are in agreement with empirical rules when using a glucose units ladder of hydrolyzed dextran (ß1-4-linked GalNAc: 0.59 gu in Wing et al., 2001Go). Based on integrated peak areas of three injections, the following percentages were found for the Sda pentasaccharide: pair of twins A, 37.7% ± 0.8% (A1) and 37.3% ± 0.8% (A2); pair of twins B, 46.8% ± 0.8% (B1) and 47.7% ± 0.8% (B2). These data clearly show identical molar ratios of Sda pentasaccharide and precursor tetrasaccharide for THp A1 and THp A2, as well as for THp B1 and THp B2.

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., 1998aGo), 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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Fig. 5. HPAEC Fractionation patterns of Superdex-75 fractions II on CarboPac PA-1 with PAD. (A) THp A1 (gray) and A2 (black) derived samples, Std = internal standard galacturonic acid (light gray); (B) THp B1 (gray) and B2 (black) derived samples, Std = internal standard galacturonic acid (light gray). For elution details, see Materials and methods. II.a: Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal; II.b: Neu5Ac({alpha}2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal.

 
Comparison of the quantitative data from the HPLC-FD and the HPAEC-PAD approaches show systematic differences, which, however, do not influence the final conclusions. In view of the type of detection used in HPAEC-PAD, absolute quantification of oligosaccharides based on peak areas should be considered with care. Different oligosaccharides can have completely different molar responses (Townsend et al., 1988Go). In the present study no molar adjustment factors for corrections were available for the individual components in the determination by HPAEC-PAD. Fluorescently labeled structures are quantified on the basis of the label absorbance, whatever the original molecule may be. This means that the values obtained by HPLC-FD can be considered as more reliable than those obtained by HPAEC-PAD.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Extensive studies on the THp glycosylation have shown an impressive panel of N- and O-glycan structures (Easton et al., 2000Go; Hård et al., 1992Go; van Rooijen et al., 1998aGo,bGo, 1999Go). The occurrence of donor specificity in male THp has been demonstrated for dimeric N-acetyllactosamine sequences with or without the Sda epitope (van Rooijen et al., 1998aGo) and for oligomannose-type N-glycans (see van Rooijen et al., 2001Go, and references cited therein). For females a pregnancy-associated variability has been reported for the total amount and type of oligomannose-type N-glycans (Smagula et al., 1990Go; van Rooijen et al., 2001Go), whereas O-glycans undergo a change from core 1-type to core 2-type (Easton et al., 2000Go). Furthermore glycosylation changes have been reported in relation to age (Goodall and Marshall, 1980Go; Reinhart et al., 1991Go), malignancy (Olczak et al., 1999Go), and renal deficiency (Storch et al., 1992Go; Torffvit et al., 1998Go).

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., 1998aGo). 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({alpha}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, 1983Go; Serafini-Cessi et al., 1986Go). ß-1,4-N-Acetylgalactosaminyltransferase activity has been demonstrated in microsomal preparations of human and guinea pig kidneys (Piller et al., 1986Go; Serafini-Cessi et al., 1986Go), whereas a soluble form was found in human colon carcinoma cells (Serafini-Cessi et al., 1995Go). 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({alpha}2-3)Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1- into the Neu5Ac({alpha}2-3)[GalNAc(ß1-4)]Gal(ß1-4)GlcNAc(ß1-3)Gal(ß1- sequence.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
General
For each of the four donors (two pairs of twins), THp was isolated from pooled morning urine as described (Serafini-Cessi et al., 1989Go), and checked for purity by SDS–PAGE and MALDI-TOF MS (matrix: 10 mg/mL {alpha}-cyano-4-hydroxy-cinnamic acid in water) (Beavis and Chait, 1996Go) using a Voyager-DE PerSeptive Biosystems instrument. Monosaccharide analysis of intact THp samples and quantification of endo-ß-galactosidase-released oligosaccharides were performed in triplicate by gas-liquid chromatography (Kamerling and Vliegenthart, 1989Go).

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., 1996Go) 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., 1992Go).

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., 1998aGo).

Fluorescent labeling of oligosaccharides with 2-AB and analysis by HPLC
Oligosaccharides were fluorescently labeled with 2-AB as described (Bigge et al., 1995Go). 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 ({lambda}exc.max = 373 nm, {lambda}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., 1996Go). Molar ratios were determined by comparison of integrated peak areas from three injections (van Rooijen et al., 1998aGo).


    Acknowledgements
 
This work was supported by the European Community research training network Glycotrain (HPRN-CT-2000-00001).


    Footnotes
 
1 To whom correspondence should be addressed; Fax: (+31)-30-2540980; e-mail: j.p.kamerling{at}chem.uu.nl


    Abbreviations
 
2-AB, 2-aminobenzamide; GPI, glycosylphosphatidyl inositol; gu, glucose unit; HPLC-FD, high-performance liquid chromatography with fluorescence detection; HPAEC-PAD, high-pH anion exchange chromatography with pulsed amperometric detection; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; NMR, nuclear magnetic resonance; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; THp, Tamm-Horsfall glycoprotein


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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