Glycosylation sites and site-specific glycosylation in human Tamm-Horsfall glycoprotein

Johannes J.M.van Rooijen, Anton F.Voskamp, Johannis P.Kamerling and Johannes F.G.Vliegenthart1

Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80075, NL-3508 TB Utrecht, The Netherlands

Received on March 2, 1998; revised on May 15, 1998; accepted on May 25, 1998

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz 1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4S([beta]1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2 to Man8GlcNAc2, in addition to a small amount of complex-type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.

Key words: Tamm-Horsfall glycoprotein/carbohydrate/NMR/site-specific glycosylation

Introduction

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.

Results

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 1 has been followed. The amino acid sequence of human THp has been published before (Hession et al., 1987; Pennica et al., 1987) and is shown in Figure 2.


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 3), and the glycopeptide-containing fractions were detected by orcinol/H2SO4, and denoted PI-PIV.


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 4a-d). The deglycosylated peptides, denoted PI2-PI7, PII11-PII14, PIII9-PIII10, and PIV4, were subjected to amino acid sequence analysis for at least five N-terminal amino acids (Diagram II). The liberated N-glycan fractions, RCPI, RCPII, RCPIII, and RCPIV, were analyzed by 500 MHz 1H-NMR spectroscopy (Figure 5a-d), in conjunction with profiling by HPAEC on CarboPac PA-100 (Figure 6b-e). In connection with the HPAEC profiling, in Figure 6a the fractionation of the total pool of N-glycans released by PNGase-F from fraction P is depicted. Here, the assignment of the regions differing in charge, Q0-Q7, is based on a sequential Resource Q-CarboPac PA-100 approach (Hermentin et al., 1996). Using the defined regions in Figure 6a, it is possible to identify the regions differing in charge (charge fractions) as indicated in Figure 6b-e. Ratios in percentage of different charge fractions of the carbohydrate pool of fractions RCPI-RCPIV, and fraction P itself are displayed in Figure 7.


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 4d). Peptide fraction PIV4 was subjected to N-terminal amino acid sequence analysis affording the partial sequence T-P-X-X-X-A-T-D-P-L-K-. As is evident from Figure 3, this sequence represents Thr485-Lys495, containing Asn489 as glycosylation site. This glycosylation site is mainly substituted with di- and tri-charged oligosaccharides as concluded from the HPAEC profile of RCPIV (Figures 6e, 7d). 1H-NMR analysis of RCPIV (Figure 5d) showed the presence of the GalNAc4S([beta]1-4)GlcNAc([beta]1-2)Man([alpha]1-3)- element in the carbohydrate moiety, as deduced from the structural-reporter-group signals of GalNAc (H-1, [delta] 4.586; H-4, [delta] 4.692; NAc, [delta] 2.069) (Hård et al., 1992; van Rooijen et al., 1998b). Furthermore, the Gal3S residue (H-3, [delta] 4.34; H-4, [delta] 4.29) (Kamerling et al., 1988; Hård et al., 1992; van Rooijen et al., 1998b), the Sda-determinant (Neu5Ac H-3a, [delta] 1.927 and H-3e, [delta] 2.662; GalNAc H-1, [delta] 4.73-4.76 (42°C) and NAc, [delta] 2.015-2.019) (Hård et al., 1992; van Rooijen et al., 1998a,b), the Neu5Ac([alpha]2-3)Gal element (Neu5Ac H-3a, [delta] 1.801 and H-3e, [delta] 2.754) (Hård et al., 1992), and the Neu5Ac([alpha]2-6)Gal element (Neu5Ac H-3a, [delta] 1.719 and H-3e, [delta] 2.668) (Hård et al., 1992) are present.

Fraction PIII (Figure 4c). 1H-NMR analysis of fraction RCPIII showed the presence of oligomannose-type glycans as the main components (Figure 5c) (Man5GlcNAc2: Man-4 H-1, [delta] 5.097; Man6GlcNAc2: Man-4 H-1, [delta] 5.344 and Man-C H-1, [delta] 5.050; Man7GlcNAc2: Man-C H-1, [delta] 5.307 and Man-D1 H-1, [delta] 5.041). This observation was supported by the HPAEC profile of RCPIII yielding 67% neutral oligosaccharides (Figures 6d, 7c) and 33% complex-type carbohydrate structures. Both peptide fractions PIII9 and PIII10 (Figure 4c) were subjected to N-terminal amino acid sequence analysis affording the partial sequences A-G-G-Y-Y- and PeC-A-G-G-Y-Y-V-Y-, respectively. As illustrated in Figure 2, the sequences of fractions PIII9 and PIII10 represent Ala244-Tyr248 and Cys243-Tyr250, respectively, meaning that both fractions were indicative for glycosylation site Asn251.

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 8a). Upon HPLC on ChromSpher 5 C8, fraction P(Con-A) released by elution with methyl [alpha]-d-mannopyranoside, comprised only one glycopeptide fraction (Figure 8b). After treatment of this fraction by PNGase-F, the digest was fractionated by HPLC on ChromSpher 5 C8 yielding subfraction RCP(Con-A) containing released glycans, and subfraction P(Con-A)1 representing deglycosylated peptide material (Figure 8c). The deglycosylated peptide P(Con-A)1 was subjected to N-terminal amino acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). The N-terminal amino acid sequence analysis afforded the partial sequence PeC-A-G-G-Y-Y-V-Y-, corresponding with the sequence Cys243-Tyr250, being indicative of a glycosylated Asn251 residue (Figure 2). The FAB-mass spectrum of fraction P(Con-A)1 contained two intense pseudomolecular ion pairs. The more intense of these, m/z 1282 [M+H]+ and 1304 [M+Na]+, corresponds in mass to the carboxymethylated peptide Cys243-Thr253 in which the glycosylated Asn251 residue has been converted into Asp251 due to the action of PNGase-F. The less intense pair of ions at m/z 993 [M+H]+ and 1015 [M+Na]+ corresponds to peptide Gly246-Thr253, a breakdown product of the major component. HPAEC profiling of the glycan mixture in fraction RCP(Con-A) (Figure 9a) indicated the presence of 6% Man5GlcNAc2, 79% Man6GlcNAc2, 13% Man7GlcNAc2, and 2% Man8GlcNAc2 (reference compounds isolated from RNase-B, Figure 9b). 1H-NMR analysis of fraction RCP(Con-A) confirmed the findings of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2. In Man7GlcNAc2 Man-C is substituted with ([alpha]1-2)Man (Man-C H-1 at [delta] 5.307; van Kuik et al., 1992). Because of the low amount of material, Man8GlcNAc2 could not be confirmed by 1H-NMR spectroscopy. Precise structures are given in Figure 9a. In conclusion, glycosylation site Asn251 contains predominantly oligomannose-type glycans, ranging from Man5GlcNAc2 to Man8GlcNAc2. Furthermore, this glycosylation site is the only one present in THp which contains oligomannose-type carbohydrate structures.

Fraction PII (Figure 4b). Peptide fractions PII12, PII13, and PII14 were subjected to N-terminal amino acid sequence analysis yielding the overlapping partial sequences N-T-A-A-P-, A-A-P-M-W-L-, and R-C-N-T-A-, respectively. As is evident from Figure 2, these sequences represent Asn200-Pro204, Ala202-Leu207, and Arg198-Ala202, respectively, indicative of glycosylation site Asn208. The N-terminal amino acid sequence analysis of the minor fraction PII11 (D-PeC-K-S-N-, Asp281-Asn285; Figure 2) is indicative of glycosylation site Asn298. The HPAEC profile of carbohydrate fraction RCPII (Figure 6c) shows the same microheterogeneity and ratio between the different charge fractions, as the HPAEC profile of the total carbohydrate moiety of THp (Figure 6a), except for the neutral carbohydrate chains (Figure 7b). 1H-NMR analysis of fraction RCPII (Figure 5b) showed the presence of Gal3S residues, the Sda-determinant, the Neu5Ac([alpha]2-3)Gal element, and the Neu5Ac([alpha]2-6)Gal element. In view of the relatively low amount of peptide PII11, correlated with Asn298, it can be concluded that the carbohydrate chains released from glycopeptide fraction PII mainly resemble the site-specific glycosylation of Asn208.


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 4a). N-Terminal amino acid sequence analysis of peptide fraction PI2 showed the partial sequence N-PeC-Y-A-T-P-, which corresponds with Asn481-Pro486 (Figure 2), therefore being indicative of glycosylation site Asn489. The peptide in fraction PI3 reflects glycosylation site Asn372, as can be deduced from the N-terminal amino acid sequence analysis demonstrating the sequence G-P-PeC-G-T-, corresponding with Gly363-Thr367 (Figure 2). Subjection of fraction PI4 to N-terminal amino acid sequence analysis afforded the partial sequence H-PeC-Q-PeC-K-, corresponding with the sequence His290-Lys294. This is indicative of a peptide containing glycosylated Asn298. N-Terminal amino acid sequence analysis of fractions PI5, PI6, and PI7 revealed partial sequences of peptides containing glycosylation sites Asn52 and Asn56 (PI5: PeC-A-I-P-G-A-H-D-C-S-A-D-S-, Cys45-Ser57; PI6: PeC-A-I-P-G-, Cys45-Gly49; PI7: PeC-A-I-P-G-, Cys45-Gly49). Both Asn52 and Asn56 are glycosylated as follows from the partial sequence of the peptide in fraction PI5, wherein Asn52 and Asn56 occur as Asp52 and Asp56, respectively. 1H-NMR analysis of the oligosaccharide fraction RCPI showed the presence of Gal3S residues, the Sda-determinant, Neu5Ac([alpha]2-3)Gal elements, and Neu5Ac([alpha]2-6)Gal elements. The HPAEC profile of carbohydrate fraction RCPI (Figure 6b) reflects a large microheterogeneity and the ratio of the different charge fractions is similar to that found for the total carbohydrate moiety of THp (Figure 7a), except for the neutral carbohydrate chains. Note that the oligosaccharides found in fraction PI represent the total carbohydrate pattern of five glycosylation sites (Asn52, Asn56, Asn298, Asn 372, and Asn489).

Discussion

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 10). The potential glycosylation site at Asn14, being close to the N-terminus, is not used. Here, also the microheterogeneity of the glycosylation sites Asn489, Asn251, and Asn208 has been analyzed in more detail.


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 4a) ranging from Man5GlcNAc2 to Man8GlcNAc2. Besides these oligomannose-type carbohydrate chains (67%), glycosylation site Asn251 contains 33% of complex-type N-glycans. The molar ratio of 6:79:13 for Man5GlcNAc2:Man6GlcNAc2:Man7GlcNAc2 is in agreement with reference data (Dall'Olio et al., 1988). However, we demonstrated the additional presence of Man8GlcNAc2 in a low amount (in the molar ratio of 2). In earlier studies THp has shown to contain 20% (Smagula et al., 1990; Serafini-Cessi et al., 1984a; based on peak heights), 16% (Dall'Olio et al., 1988), 2% (Afonso et al., 1981), and 0% (Hård et al., 1992) oligomannose-type carbohydrate chains. The level of oligomannose-type carbohydrate chains is therefore possibly a donor-specific feature. It should be noted that oligomannose-type carbohydrate chains are supposed to play a predominant role in the immunosuppressive properties displayed by THp (Serafini-Cessi et al., 1979; Muchmore et al., 1987, 1990a,b; Moonen et al., 1988; Smagula et al., 1990).

Finally, glycosylation site Asn208 shows a large microheterogeneity resembling that of the total glycan pool of THp (Figures 7a and 7c).

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 and methods

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.

Acknowledgments

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.).

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