Human Tamm-Horsfall glycoprotein (THp) is the most abundant protein in normal human urine, being excreted in quantities of up to 100 mg/day (Tamm and Horsfall, 1950; Tamm and Horsfall, 1952). THp is produced by the kidney where it is expressed via a phosphatidylinositol anchor on the endothelium of the thick ascending limb of the loop of Henle (Sikri et al., 1979; Rindler et al., 1990). The physiological function of THp is still controversial. THp could act as a natural inhibitor of microbial infection of the urinary tract and urinary bladder (Duncan, 1988; Parkkinen et al., 1988). A possible relation between THp and the electrolyte/water transport in the nephron has been described previously (Mattey and Naftalin, 1992) as well as a role in the formation of kidney stones (Hallson and Rose, 1979; Scurr and Robertson, 1986). The ability to inhibit antigen-specific T-cell proliferation in vitro probably due to the glycan part of the protein has been reported but the results are ambiguous (Muchmore et al., 1987; Moonen et al., 1988). Furthermore, the carbohydrate chains of THp have been implicated in the binding to neutrophils (Toma et al., 1994) and in immunosuppressive properties (Sathyamoorthy et al., 1991).
In earlier studies, it has been found that THp built up from a polypeptide backbone of 616 amino acids, has a carbohydrate content of 25-30% (w/w) (Kumar and Muchmore, 1990), distributed over eight potential N-glycosylation sites, Asn14, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489 (Hession et al., 1987; Pennica et al., 1987), of which at least five are occupied (Afonso et al., 1981). So far, no data have been reported about the specific asparagine residues involved in the glycosylation, nor about the heterogeneity of the carbohydrate chains at each occupied glycosylation site.
Detailed structural studies of the total glycosylation pattern of THp from individual male donors have resulted in the elucidation of 63 complex-type N-glycans, which form only part of the 150 isolated carbohydrate-containing fractions (Hård et al., 1992; van Rooijen et al., 1998a, b; see also Donald et al., 1983; Serafini-Cessi et al., 1984b; Williams et al., 1984; Donald and Feeney, 1986). Di-, tri-, and, most of all, tetraantennary structures (including dimeric N-acetyllactosamine sequences) are present which can be fucosylated, sialylated (including the Sda-determinant) and/or sulfated. Furthermore, oligomannose-type carbohydrate chains are reported for THp from pooled urine of various individuals ranging from Man5GlcNAc2 to Man7GlcNAc2 (Serafini-Cessi et al., 1984a; Dall'Olio et al., 1988; Smagula et al., 1990).
In this study, a detailed analysis of the glycosylation sites of THp is presented in order to contribute to the unraveling of the biological functions of human THp.
THp (100 mg) was isolated from 2 l morning urine from a healthy male donor. It was checked for purity by SDS-PAGE, demonstrating a single band at 94 kDa under reducing conditions. Monosaccharide analysis of THp revealed the presence of Fuc, Man, Gal, GalNAc, GlcNAc, and Neu5Ac in a molar ratio of 0.9:3.0:3.7:1.1:5.5:2.9, and the carbohydrate content was found to be 28% by mass. In order to generate information with respect to glycosylation sites and site-specific glycosylations in THp, the analysis strategy as depicted in Figure
Figure 1. Work-up procedure for the V-8-protease digest of reduced and S-carboxymethylated THp.
Figure 2. The amino acid sequence of THp, as deduced from cDNA and chemical sequencing (Hession et al., 1987; Pennica et al., 1987). The Asn residues of the potential N-glycosylation sites are written in boldface type. Arrow through PII12 represents the N-terminal amino acid sequence of peptide PII12 generated by PNGase-F treatment of glycopeptide fraction PII.
After reduction and S-carboxymethylation, THp (50 mg) was incubated with endoproteinase Glu-C (V-8 protease), and the progress of digestion was followed by HPLC on ChromSpher 5 C8. Gel permeation chromatography on HiTrap of the digest afforded fraction P, containing the high-molecular-mass (glyco)peptides. This fraction was subjected to HPLC on ChromSpher 5 C8 (Figure
Figure 3. Fractionation pattern at 220 nm on ChromSpher 5 C8 of (glyco)peptide fraction P. The elution was carried out on a ChromSpher 5 C8 column (10 × 250 mm, Chrompack), at a flow rate of 2.5 ml/min. The column was eluted isocratically with solvent A (aqueous 5% acetonitrile containing 0.1% trifluoroacetic acid) for 5 min, followed by a linear concentration gradient from 100% solvent A/0% solvent B (aqueous 80% acetonitrile containing 0.1% trifluoroacetic acid) to 43% solvent A/57% solvent B in 55 min.
In order to characterize which of the potential glycosylation sites are glycosylated, fractions PI, PII, PIII, and PIV were each treated with PNGase-F and then separated by HPLC on ChromSpher 5 C8 (Figure
Figure 4. Elution profiles of PNGase-F-treated glycopeptide fractions isolated from fraction P. (a) fraction PI, (b) fraction PII, (c) fraction PIII, and (d) fraction PIV. Elutions were performed as described in Figure 3. Fraction RCPI, RCPII, RCPIII, and RCPIV contain the carbohydrate chains liberated from the peptide fractions PI2-PI7, PII11-PII14, PIII9-PIII10, and PIV4, respectively.
Figure 5. Resolution-enhanced 500 MHz 1H-NMR spectra at 300 K of fraction (a) RCPI, (b) RCPII, (c) RCPIII, and (d) RCPIV. Structural elements are displayed at the position of their structural-reporter-group signals (Hård et al., 1992; van Rooijen et al., 1998a,b).
Figure 6. HPAEC profiles of the released carbohydrate chains of the PNGase-F-treated glycopeptide fractions PI-PIV on CarboPac PA-100 with pulsed amperometric detection. Elutions were carried out on a CarboPac PA-100 column (4.6 × 250 mm, Dionex) with a concentration gradient of NaOAc in 0.1 M NaOH as indicated in (a), at a flow rate of 1 ml/min. The assignment of the regions differing in charge, charge fractions Q0-Q7, is based on a sequential Resource Q-CarboPac PA-100 approach (Hermentin et al., 1996). (a) total carbohydrate pool of P; (b) RCPI; (c) RCPII; (d) RCPIII; and (e) RCPIV.
Fraction PIV (Figure
Fraction PIII (Figure
In order to investigate if the presence of oligomannose-type structures is restricted to only one glycosylation site in THp, fraction P (5 mg) was subjected to ConA Sepharose affinity chromatography (Figure
Fraction PII (Figure
Figure 7. The relative ratio (%) of charge fractions Q0-Q7 calculated from the HPAEC profiles of the released carbohydrate chains of peptide fractions PI, PII, PIII, and PIV displayed in Figure 6. (a) RCPI; (b) RCPII; (c) RCPIII; (d) RCPIV. Plotted line represents the relative ratio (%) of charge fractions Q0-Q7 of the total carbohydrate moiety obtained from PNGase-F-treated fraction P.
Figure 8. (a) Elution profile at 206 nm on ConA-Sepharose (Pharmacia) of (glyco)peptide fraction P. The elution was started with 30 ml 20 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl, 1 mM CaCl2, and 1 mM MgCl2 (Buffer A) at a flow rate of 15 ml/h. Then, elution of retained glycopeptides containing diantennary complex- and oligomannose-type oligosaccharides was performed by eluting with 25 ml 10 mM methyl [alpha]-d-glucopyranoside in buffer A at room temperature (start is indicated in the figure as A) and 30 ml 100 mM methyl [alpha]-d-mannopyranoside in buffer A at 60°C (start is indicated in the figure as B), respectively. P(Con-A) represents the fraction released by methyl [alpha]-d-mannopyranoside. (b) Elution profile at 220 nm on ChromSpher 5 C8 of fraction P(Con-A). Conditions are described in Figure 3. (c) Elution profile at 220 nm on ChromSpher 5 C8 of PNGase-F-treated fraction P(Con-A). RCP(Con-A) contains the liberated oligosaccharides from the deglycosylated peptide P(Con-A)1. Conditions are described in Figure 3.
Figure 9. HPAEC profiles of oligomannose-type carbohydrate chains on CarboPac PA-100 with pulsed amperometric detection. (a) Fraction RCP(Con-A), containing the released oligosaccharides of the PNGase-F-treated glycopeptide fraction P(Con-A). (b) Oligomannose-type structures of RNase-B. M5 to M9 represent oligomannose-type structures Man5GlcNAc2 to Man9GlcNAc2, respectively. Compounds are represented by shorthand symbolic notation: solid diamonds, Man; solid circles, GlcNAc. For Man9GlcNAc2 the numbering of the individual monosaccharide residues is given. Elutions were performed as indicated in the figure.
Fraction PI (Figure
Tamm-Horsfall glycoprotein has previously been shown to contain eight potential glycosylation sites (Hession et al., 1987; Pennica et al., 1987), of which at least five were glycosylated (Afonso et al., 1981). In this study, we characterized the glycosylation sites of THp from one healthy male donor by analysis of glycopeptides formed by proteolysis by V-8 protease. In some cases, more than one glycopeptide was generated representing the same glycosylation site. Out of the eight potential glycosylation sites, seven are occupied, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489 (Figure
Figure 10. Schematic representation of THp including its specific domains and potential glycosylation sites. THp contains eight potential glycosylation sites, but only Asn14 is not glycosylated. Three EGF-like domains and one ZP-domain are present (Hession et al., 1987; Pennica et al., 1987; SWISS-PROT Database, id. p07911) as indicated in the figure. EGF-like domain, epidermal growth factor-like domain; ZP-domain, zona pellucida domain.
It should be noted that in view of the complexity of the structural studies of THp, so far no quantitative data with respect to the level of site-occupancy of the seven Asn residues has been generated.
Glycans at Asn489 can be terminated by GalNAc4S([beta]1-4)GlcNAc and mainly di- and tri-charged N-glycans are observed. These findings are in agreement with the observation that the GalNAc4S([beta]1-4)GlcNAc structural element is only established yet for diantennary N-glycans of THp (compounds N3.2.1 and N3.2.3 in Hård et al., 1992; compounds Q3.1.1, Q3.1.3, Q3.1.5 and Q3.1.11B in van Rooijen et al., 1998b), although evidence was found for GalNAc4S-containing triantennary compounds as well (van Rooijen et al., 1998b). Other urinary glycoproteins, like urokinase (Bergwerff et al., 1995) and kallidinogenase (Tomiya et al., 1993), also display the GalNAc([beta]1-4)GlcNAc element. In the case of urokinase also 4-O-sulfated GalNAc occurs. Interestingly, it should be noticed that the GalNAc4S([beta]1-4)GlcNAc structural element seems to occur exclusively at Asn489 with a Pro-Leu-Lys peptide motif, located 3 residues on the COOH-terminal side. So far, it has been suggested that the sequon Pro-Xaa-Arg/Lys at the NH2-terminal side at various positions is responsible for directing the specificity of the [beta]4-GalNAc-transferase (Smith and Baenziger, 1992; Sato et al., 1995). Since such a sequon is located now also at the COOH-terminal direction of the Asn glycosylation site, it may be rather the influence of the three-dimensional structure of the protein near the glycosylation site that is responsible than a sequon.
Only glycosylation site Asn251 contains oligomannose-type structures (5% of the total carbohydrate pool based on the ratios on CarboPac PA-100, Figure
Finally, glycosylation site Asn208 shows a large microheterogeneity resembling that of the total glycan pool of THp (Figures
The detailed knowledge of the glycosylation pattern of THp including the donor-specificity (Hård et al., 1992; van Rooijen et al., 1998a,b; this study), makes further studies with respect to the biological significance of THp challenging.
Materials
THp was isolated from pooled morning urine of a healthy male donor as described previously (Serafini-Cessi et al., 1989). Recombinant peptide-N4-(N-acetyl-[beta]-glucosaminyl)asparagine amidase F (PNGase-F) from Flavobacterium meningosepticum (EC 3.5.1.52) and endoproteinase Glu-C (V-8 protease, EC 3.4.21.1g) from Staphylococcus aureus were purchased from Boehringer Mannheim, Germany.
Reduction and S-carboxymethylation
THp was reduced and S-carboxymethylated using standard methods (Lustbader et al., 1989). Briefly, THp (75 mg) was dissolved in 20 ml 1 M Tris-HCl, pH 8.25, containing 6 M guanidine-HCl, 1 mM EDTA and 50 mM DTT, and the mixture was incubated for 2 h at 37°C. After cooling down to room temperature, iodoacetic acid (0.5 M in 0.5 M NaOH) was added to a final concentration of 100 mM, and the mixture was incubated in the dark for 30 min. The reaction was quenched by adding an excess of [beta]-mercaptoethanol. Salts were removed by dialyzing the sample three times against water. Next, the THp solution was lyophilized and stored at -20°C prior to use. The purity was checked by SDS-PAGE under reducing and nonreducing conditions.
Endoproteinase Glu-C digestion
Reduced and carboxymethylated THp (50 mg) was dissolved in 15 ml 50 mM phosphate buffer, pH 7.8, and incubated with endoproteinase Glu-C (1 mg) at 37°C. After 18 h, a second portion of endoproteinase Glu-C (1 mg) was added and the digestion was continued for 18 h at 37°C. After lyophilization, the digest was fractionated on four connected HiTrap columns (4 × 5 ml, Pharmacia) using 5 mM NH4HCO3, pH 7.0, as eluent at a flow rate of 4 ml/min. The effluent was monitored at 278 nm and the void-volume fraction, containing the glycopeptides (orcinol/H2SO4 assay), was isolated and lyophilized.
Affinity chromatography
An aliquot of the glycopeptide mixture (5 mg) was applied to a Concanavalin A Sepharose column (2 ml, Pharmacia) and first eluted with 30 ml 20 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl, 1 mM CaCl2, and 1 mM MgCl2 (buffer A) at a flow rate of 15 ml/h. Then the elution was continued with 25 ml 10 mM methyl [alpha]-d-glucopyranoside in buffer A at room temperature to desorb diantennary-complex-type glycopeptides, and finally with 30 ml 100 mM methyl [alpha]-d-mannopyranoside in buffer A at 60°C to desorb oligomannose-type glycopeptides. All fractions were lyophilized, desalted by HiTrap (Pharmacia FPLC system; four columns connected, 4 × 5 ml; eluent, 5 mM NH4HCO3; flow rate, 3 ml/min; detection, 206 nm), and lyophilized again. Glycopeptides containing oligomannose-type structures were fractionated by HPLC as described below.
HPLC
(Glyco)peptide mixtures were fractionated by HPLC on a ChromSpher 5 C8 column (10×250 mm, Chrompack) at a flow rate of 2.5 ml/min, using a Kratos SF 400 HPLC system (ABI Analytical, Kratos Division). The column was eluted isocratically with solvent A (aqueous 5% acetonitrile containing 0.1% trifluoroacetic acid) for 5 min, followed by a linear concentration gradient from 100% solvent A/0% solvent B (aqueous 80% acetonitrile containing 0.1% trifluoroacetic acid) to 43% solvent A/57% solvent B in 55 min. The effluent was monitored at 220 nm using a Spectroflow 757 absorbance detector (ABI Analytical, Kratos Division). Fractions were collected manually and immediately dried in a SpeedVac and lyophilized. Fractions containing glycopeptides were detected using orcinol/H2SO4.
Liberation of the N-linked carbohydrate chains from glycopeptides
Glycopeptide mixtures were dissolved in 100 µl 50 mM phosphate buffer, pH 8.2, containing 10 mM EDTA, then incubated with 0.5 U PNGase-F at 37°C. After 18 h, the digest was boiled for 3 min and cooled down to room temperature. In each case the digest was fractionated by HPLC as described above. The HPLC-void-volume fraction, containing the liberated carbohydrate chains, was desalted by HiTrap (Pharmacia), and lyophilized. The fractions containing deglycosylated peptides were immediately dried in a SpeedVac, lyophilized, and subsequently analyzed for their N-terminal amino acid sequence.
Oligosaccharide profiling on HPAEC
Each HPLC-void-volume fraction, stemming from PNGase-F-treated glycopeptides, was analyzed for oligosaccharide components by HPAEC, using a Dionex DX500 chromatography system, equipped with a CarboPac PA-100 column (4.6 × 250 mm, Dionex) (Hermentin et al., 1992). Oligosaccharides were separated using a concentration gradient of NaOAc in 0.1 M NaOH as indicated in the figures, at a flow rate of 1 ml/min. Pulsed amperometric detection was performed using the following pulse potentials and durations: E1 = 0.05 V (400 ms); E2 = 0.75 V (200 ms); E3 = -0.15 V (400 ms).
1H-NMR spectroscopy
Prior to 1H-NMR analysis, samples were exchanged twice in 99.9% 2H2O with intermediate lyophilization and finally dissolved in 450 µl 99.96% 2H2O (Isotec Inc). 500 MHz 1H-NMR spectra were recorded on a Bruker AMX-500 spectrometer (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University) essentially as described previously (Hård et al., 1992).
Fast atom bombardment mass spectrometry
Positive-ion fast atom bombardment mass spectrometry (FAB-MS) of a deglycosylated peptide sample was performed using MS1 of a JEOL JMS-SX/SX102A tandem mass spectrometer (Bijvoet Center, Department of Mass Spectrometry, Utrecht University), using 10 kV accelerating voltage. The FAB gun was operated at an emission current of 10 mA, with Xe as bombarding gas. The spectra were scanned at a speed of 30 s for full mass range specified by the accelerating voltage used, and were recorded and averaged on a Hewlett Packard HP9000 data system. Samples were dissolved in 10 µl aqueous 5% HOAc and 1 µl was loaded into a matrix of thioglycerol.
We are grateful to Dr. J. Thomas-Oates (Bijvoet Center, Department of Mass Spectroscopy) for recording and interpreting the FAB-mass spectrum. This work was supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for Scientific Research (N.W.O.).
1To whom correspondence should be addressed