Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl*

O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed byN-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

Thrombomodulin (TM) 1 is a physiologically important anticoagulant (1) that is present not only on the endothelial cell surface but also in soluble form in plasma and urine (2)(3)(4). Takahashi et al. (5) have purified the major active forms of human urinary soluble thrombomodulin (uTM) and demonstrated that they possess strong cofactor activity for thrombincatalyzed protein C activation as well as exhibiting potent anticoagulant activity in vivo. They have also shown that uTM improves disseminated intravascular coagulation without excessive prolongation of the activated partial thromboplastin time (6). The protein possesses five potential N-linked glycosylation sites (7) as deduced from its amino acid sequence, whereas the detection of GalNAc suggests the presence of Olinked sugar chains (8). Although uTM does not contain a glycosaminoglycan, recombinant TM and some TMs obtained from cultured human endothelial cells are expressed in both a high molecular weight TM containing chondroitin sulfate and a low molecular weight TM lacking this modification (9,10). Recombinant TM expressed in Chinese hamster ovary cells contains chondroitin 4-sulfate (11). TM glycosylation in relation to biological activity has been discussed in several papers (12)(13)(14), but to understand the structure and biological activities of the glycoprotein, detailed knowledge of the sugar structures is essential. Recently, the structures of N-linked sugar chains have been reported (15). During the course of studies on N-and O-linked sugar chains of uTM, we have detected new structures with xylose at the reducing ends. Here, we present a detailed analysis of the novel sugar structures found in uTM.
Preparation of Glycopeptides-uTM (374 mg) was digested with 37 mg of actinase E in 3.7 ml of 0.2 M Tris-HCl buffer, pH 8.2, at 37°C for 1 h. An additional 3.7 mg of the enzyme was then added, and the incubation was continued for 24 h. The digest was applied onto a Toyopearl HW-40F column (2.2 ϫ 100 cm), and glycopeptides were eluted with 0.2 M ammonium acetate buffer, pH 6.0.
Preparation of PA Sugar Chains-PA sugar chains were prepared and purified according to the reported method (18). A freeze-dried glycopeptide fraction was heated with 1.6 ml of anhydrous hydrazine at 60°C for 50 h followed by N-acetylation with 3.2 ml of saturated sodium bicarbonate solution and 128 l of acetic anhydride. After desalting with Dowex 50 (H ϩ ), the solution was freeze-dried, and the residue was pyridylaminated with 160 l of 2-aminopyridine reagent and 560 l of reducing reagents prepared as reported (18). Excess reagents were evaporated three times with 50 l of toluene:methanol (1:1) under a stream of nitrogen at 60°C for 10 min.
HPLC of PA Sugar Chains-HPLC was carried out using the following elution conditions. PA sugar chains were detected by fluorescence (excitation wavelength, 320 nm; emission wavelength, 400 nm).
Elution Condition 1 for PA O-linked sugar chains: column, Cosmosil 5C18-AR300; flow rate, 2 ml/min at 25°C; eluent, 20 mM ammonium acetate buffer, pH 6.0, containing 0.01% 1-butanol. After injecting a sample, the butanol concentration was increased linearly from 0.01 to 0.52% in 51 min and then to 1.0% in 12 min. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Pharmaceutical Laboratory, Mocida Pharmaceutical Co., Ltd., Fujieda, Shizuoka 426-8640, Japan.
Anion-exchange HPLC: column, TSKgel Sugar AXI; flow rate, 0.3 ml/min at 73°C. The eluent used was a mixture of 9 parts 0.8 M boric acid adjusted to pH 9.0 with potassium hydroxide and 1 part acetonitrile.
Mono Q HPLC: column, Mono Q HR 5/5; flow rate 1.0 ml/min at 25°C. The eluent used was water titrated to pH 9.0 with aqueous ammonia. Adsorbed samples were eluted with a linear gradient of ammonium sulfate from 0 to 1.0 M concentration. Exoglycosidase Digestion-A PA sugar chain (100 pmol) was digested with 100 milliunits of Aspergillus ␤-galactosidase in 20 l of 50 mM ammonium acetate buffer, pH 4.5; with 2.5 milliunits of sialidase in 50 l of 100 mM ammonium acetate buffer, pH 5.0; or with 100 units of ␤-glucuronidase in 100 l of 200 mM ammonium acetate buffer, pH 5.0. Enzymatic reactions were carried out at 37°C for 16 h and then terminated by heating at 100°C for 3 min.
Methanolysis-A PA sugar chain (500 pmol) was methanolyzed with 50 mM HCl in methanol at 37°C for 3 h (19). After evaporation to dryness, the desulfated PA sugar chain was N-acetylated with 120 l of water:pyridine:acetic anhydride (5:25:1) on ice for 1 h. After evaporation of the solution, a small amount of O-acetyl groups was removed by heating the solution with 20 l of 1.0 M aqueous ammonia at 100°C for 5 min. The solution was then freeze-dried.
Methylation Analysis-Freeze-dried X2 (2 nmol) was permethylated with 0.1 ml of methylsulfinyl carbanion reagent (prepared from 25 mg of NaH and 0.8 ml of dimethyl sulfoxide) and 0.1 ml of methyl iodide as reported (20). The reaction mixture was placed on a Sephadex LH 20 column (0.9 ϫ 24 cm), and the permethylated X2 was eluted with chloroform. One-ml fractions were collected, and each fraction was concentrated to dryness by blowing the solvent with nitrogen. The residue was dissolved in a small amount of chloroform, and the sample was purified by TLC using a silica gel plate and methanol:ethyl acetate (2:8, v/v) with one drop of acetic acid as a solvent. The fluorescent spots revealed under a UV lamp were scraped and combined, and the permethylated X2 was extracted with 2 ml of methanol. The extract was evaporated to dryness, and the permethylated X2 thus purified was then hydrolyzed with 50 l of 4 M trifluoroacetic acid at 100°C for 3 h. The solution was freeze-dried, and the residue was pyridylaminated with 10 l of 2-aminopyridine reagent and 35 l of boranedimethylamine complex reagent as reported (21). Excess reagents were evaporated three times with 50 l of toluene:methanol (1:1 v/v) under a stream of nitrogen at 60°C for 10 min (18). The residue was dissolved in 5 l of the electrophoresis buffer, and small amounts of contaminating materials were removed by paper electrophoresis. Paper electrophoresis was performed at 30 V/cm at 4°C using a filter paper (30 cm) and water:acetic acid:pyridine (60:2:3, v/v). The area that migrated like PA GlcNAc was cut off, and PA derivatives of partially O-methylated PA monosaccharides were extracted from the paper 4 times each with 100 l of water. A part of the solution (10 l) was analyzed by reversedphase HPLC using Elution Condition 2.
Mass Spectrometric Analysis-Mass spectra were recorded using a Voyager-DE STR BioSpectrometry Workstation, a matrix-assisted laser desorption ionization time of flight mass spectrometer (PerSeptive Biosystems, Framingham, MA). For mass spectrometry, PA sugar chains were dissolved in distilled water (10 pmol/l). Aliquots of 0.5 l were applied onto a sample plate. Subsequently, 0.5 l of a matrix solution (10 mg/ml 2, 5-dihydroxybenzoic acid in 50% (v/v) acetonitrile) was mixed with the aliquot and allowed to dry. The analyzer was used in the linear mode.
Nuclear Equilibrium Dialysis-A commercial HNK-1 monoclonal antibody was purified by gel filtration on a YMC-Pack Diol-200G column, and the IgM fraction was concentrated by ultrafiltration with an Amicon YM-30 membrane. Equilibrium dialysis was performed between 60 l each of an antibody solution and an oligosaccharide solution using a Spectrapor membrane at 2°C for 48 h (22). Three concentrations of X1 (160, 80, and 40 nM) and one concentration of X2 (80 nM) were used. PA GlcNAc was employed as an internal standard. After the dialysis, a part of the oligosaccharide fraction was removed, and the amounts of X1 and X2 were quantified by size fractionation HPLC. The binding constant was obtained by double-reciprocal plots of the concentration of the bound oligosaccharide versus that of the free oligosaccharide, as reported (23).
Other Analytical Methods-The structures of the PA sugar chains were assessed by two-dimensional sugar mapping. A PA sugar chain was chromatographed by reversed-phase (Elution Condition 1) and size fractionation HPLC, and its elution position was compared with those of standard PA sugar chains on a two-dimensional sugar map. The PA sugar chain was then digested sequentially with exoglycosidases, and the structure of the product was analyzed on the two-dimensional sugar map as reported (23).  The reducing ends of PA sugar chains were analyzed according to the reported method (24). PA sugar chains were hydrolyzed with 100 l of 4 M trifluoroacetic acid at 100°C for 3 h in evacuated sealed tubes. The solution was evaporated to dryness by a centrifugal concentrator, and the residue was N-acetylated with saturated sodium bicarbonate solution and acetic anhydride. The PA monosaccharides obtained were separated and quantified by anion-exchange HPLC.
Component sugar analysis was done as reported (24). Glycopeptides were hydrolyzed with 100 l of 4 M trifluoroacetic acid at 100°C for 3 h. The solution was freeze-dried, and monosaccharides liberated were N-acetylated with saturated sodium bicarbonate solution and acetic anhydride. After desalting with Dowex 50 (H ϩ ), the solution was freezedried, and the monosaccharides were pyridylaminated with 2-aminopyridine and borane-dimethylamine complex. The excess reagents were removed under a stream of nitrogen gas with 40 l of toluene at 40°C. The residue was dissolved in a small amount of water, and a part of the solution was analyzed by anion-exchange HPLC.
Sulfate ion was measured by ion chromatography. A PA sugar chain (2 nmol) was mixed with 20 l of 20 mM sodium hydroxide. After heating at 250°C for 30 min, the resulting sulfate ion was measured with a Dionex 20210i ion chromatography system using an IonPac AS4A column and 2.8 mM Na 2 CO 3 , 2.25 mM NaHCO 3 at a flow rate of 1.5 ml/min (25). Sialic acid was measured by the reported method (26).

Preparation and Analysis of a PA Sugar Chain Fraction from
uTM-Glycopeptides were prepared from 374 mg of uTM by digestion with actinase E, and the digest was purified by gel filtration. After component analysis of each fraction, the fractions that contained sugars were combined (data not shown). Sugar chains were liberated from the combined fraction by hydrazinolysis followed by N-acetylation, and the reducing ends of the sugar chains were pyridylaminated. To obtain an overall view of the sugar structures, the reducing ends of the PA sugar chain fraction were first analyzed. The acid hydrolysates of the PA sugar chain fraction were found to contain 0.39 mol of PA Xyl, 3.0 mol pf PA GlcNAc, 0.45 mol of PA GalNAc, and 0.39 mol of PA Gal (from a by-product obtained from Gal-GalNAc structures during the preparation (18)), indicating that a sugar chain containing Xyl at the reducing end comprised about 8% (mol/mol) of the total sugar chains. Detection of PA Xyl indicated the presence of proteoglycan-type sugar chains; however, the linking of chondroitin sulfate chains was excluded as judged from the finding that the uTM bands on SDS-polyacrylamide electrophoresis gels were not changed by digestion with chondroitinase ABC (data not shown). The PA sugar chain fraction was digested with sialidase and analyzed by Mono Q HPLC, and a pass-through and adsorbed fractions were obtained. Reducing-end analysis of each fraction revealed that PA Xyl was present only in the acid hydrolysates of the adsorbed fraction, indicating that the xylose-containing sugar chains carried a negative charge(s) in addition to sialic acid (data not shown).
Purification of PA Sugar Chains with Xylose at the Reducing Ends-The PA sugar chain fraction was applied onto a TSKgel HW-40F column, and an aliquot of each fraction was subjected to reducing-end PA monosaccharide analysis (Fig. 1). A fraction containing PA Xyl (Fraction X) was collected and further fractionated by reversed-phase HPLC (Fig. 2). Reducing-end analysis of each peak showed that fractions X1 and X2 contained a PA Xyl residue as a major component. The other fractions and the fraction eluted between 25 and 60 ml did not contain appreciable amounts of PA Xyl. Other major peaks contained PA GalNAc, PA Gal, or PA GlcNAc as major components. Small amounts of contaminating materials were removed by size fractionation HPLC, and the final fractions were desalted on a Sephadex G-25 column (1 ϫ 10 cm) using 10 mM ammonium acetate, pH 6.0, as an eluent. Starting from Fraction X, X1, and X2 were recovered at 17 and 26% (including losses during purification), respectively. X1 and X2 thus purified showed a single peak when analyzed by reversed-phase HPLC (data not shown) using Elution Condition 1 and size fractionation HPLC, indicating that X1 and X2 were pure.
The above findings were confirmed by measuring the molecular weight of X1 by mass spectrometry (Fig. 4). In the positiveion mass spectrum, the peak of the molecule-related ion, [M ϩ Na] ϩ (m/z 831.2, calculated 831.2), was observed. Loss of sodium sulfite with hydrogen transfer from the cation also oc-  (29). Code numbers of sugar residues are indicated in brackets as described above. X1 was measured at 50°C, X2 at 50 and 30°C, R1 at 15°C, R2 at 22°C, and R3 at 27°C. curred, resulting in an intense peak at m/z 729.2 (calculated 729.3). In the negative-ion mass spectrum also, the results verified the proposed structures of X1. The linkage position of the sulfate group was analyzed by 1 H NMR spectroscopy. Reported data on the unsaturated linkage hexasaccharide of proteoglycans (⌬GlcA␤1-3GalNAc␤1-4GlcA␤1-3Gal␤1-3Gal␤1-4Xylol) (27), the synthetic trisaccharide, YM677 (SO 4 -3GlcA␤1-3Gal␤1-4GlcNAc) (28), and an oviducal mucin oligosaccharide (SO 4 -3GlcA␤1-3Gal␤1-4Gal␤1-3GalNAcol) (29) provided the necessary reference data for assignment of the proton signals of X1 (Table II). The proton signal at 4.605 ppm was assigned to H1 of Gal-2 and that at 4.668 ppm, to H1 of Gal-3 as compared with those of the unsaturated linkage hexasaccharide (R1). Analysis of the two-dimensional NMR spectra of the SO 4 -GlcA residues in X1 (Fig. 5) revealed identical spin patterns to the SO 4 -3GlcA residues in the the synthetic trisaccharide, YM677 (R2) and oviducal mucin oligosaccharide (R3). Therefore, the proton signals at 4.757 and 4.317 ppm were, respectively, assigned to H1 and H3 of SO 4 -3GlcA, confirming that the HNK-1 disaccharide element was present in X1. Taking these results together with those given above, the structure of X1 was determined to be SO 4 -3GlcA␤1-3Gal␤1-3Gal␤1-4Xyl-PA. Structure Analysis of X2-Partial acid hydrolysis of X2 gave the same fluorescent fragments as found for X1, indicating that X2 also contained the linkage tetrasaccharide structure. X2 had 1 mol each of a sulfate and Neu5Ac (Table I). The sialidase digest of X2 was eluted at the position of X1 on the twodimensional sugar map (Fig. 3), indicating that X2 was a sialylated form of X1. The elution position of X2 on the map was also not changed by digestion with ␤-glucuronidase, chondro-4-sulfatase, or chondro-6-sulfatase (data not shown). When X2 was desulfated by methanolysis, the product appeared at position A (Fig. 3). Desulfated X2 was then susceptible to ␤-glucuronidase digestion, and the digest was eluted at position B. These results indicated that the sulfate group was linked to the GlcA-Gal structure. On further digestion of the ␤-glucuronidase digest with Aspergillus ␤-galactosidase, a new peak with a molecular size 0.9 glucose units smaller appeared at position C. The sialidase digest of the ␤-galactosidase digest was eluted at the position of Gal␤1-4Xyl-PA, indicating that Neu5Ac was linked to the Gal-Xyl structure (Fig. 3). Methylation analysis of X2 indicated 3,6-disubstituted and 3-substituted Gal structures (Fig. 6). These results suggested that X2 was SO 4 -GlcA␤1-3Gal␤1-3(Neu5Ac␣2-6)Gal␤1-4Xyl-PA. The structure was confirmed by measuring the molecular weight of X2 by mass spectrometry (Fig. 4). In the positive-ion mass spectrum, peaks of the molecule-related ions, [M ϩ Na] ϩ (m/z 1122.2, calculated 1122.3) and [M ϩ K] ϩ (m/z 1138.5, calculated 1138.3), were observed. Loss of sodium sulfite with hydrogen transfer from these cations also occurred, resulting in an intense peak at m/z 1020.2 (calculated 1020.4). In the negative-ion mass spectrum also, the results verified the proposed structure of X2. The linkage position of the sulfate group was analyzed by 1 H NMR in the same manner as for X1. Analysis of the two-dimensional NMR spectra of the SO 4 -GlcA residue in X2 (Fig. 5) revealed identical spin patterns to the SO 4 -3GlcA residues in X1. Therefore, the proton signal at 4.756 ppm and that at 4.320 ppm were, respectively, assigned to H1 and H3 of SO 4 -3GlcA, confirming that the HNK-1 disaccharide element was also present in X2. Three typical signals (H3e 2.688, H3a 1.634, and NAc 2.012 ppm) of the Neu5Ac residue were observed. The NMR spectra of X2 were almost the same as those of X1 besides Neu5Ac and H4 of Gal-2. The H4 signal of Gal-2 in X1 was shifted, indicating that Neu5Ac was linked to C6 of Gal-2. Taking these results together with those given above, the structure of X2 is SO 4 -3GlcA␤1-3Gal␤1-3(Neu5Ac␣2-6)Gal␤1-4Xyl-PA.
Binding Analysis of HNK-1 Antibody to X1 and X2-Doublereciprocal plots of the concentrations of the bound oligosaccharide (X1) versus those of the free oligosaccharide in equilibrium dialysis gave the binding constant of HNK-1 antibody to X1. The apparent association constant was 2.5 ϫ 10 6 M Ϫ1 . The binding constant of X2, 1 ϫ 10 6 M Ϫ1 , was smaller than that of X1. These analyses showed that Neu5Ac in X2 did not contribute to the binding to the antibody used.

DISCUSSION
Analysis of sugar chains from human uTM revealed the presence of oligosaccharides with xylose at the reducing end. On the basis of the results obtained with X1 and X2, the structures were determined to be linkage tetrasaccharides of proteoglycans with SO 4 -3GlcA at the nonreducing end and with partial substitution of sialic acid. These are novel structures and to our knowledge are the first reported instance of a proteoglycan linkage tetrasaccharide with SO 4 -3GlcA and sialic acid residues solely linked to a glycoprotein. Because X1 and X2 were liberated and their structures determined by chemical means, the possibility of another chemically labile substituent(s) cannot be excluded. uTM does not contain chondroitin sulfate (2)(3)(4), although TM produced in cultured human endothelial cells and recombinant TMs were expressed in both cases with a high molecular weight TM containing chondroitin sulfate and a low molecular weight TM lacking this modification (10). Platelet factor 4 binds to the glycanated form of TM but not to the chondroitin sulfate-lacking TM (12). Acceleration of the inhibition of thrombin by antithrombin III by TM is dependent upon the presence of chondroitin sulfate linked to TM (10). Hence, the binding of chondroitin sulfate seems to be important for several of the versatile functions of TM and to affect its cell-surface anticoagulant potential. Chondroitin sulfate is linked to Ser-474 in the high molecular weight TM of recombinant TM, but Ser-474 is also modified with a small substituent in the low molecular weight TM (9,10). By way of explanation of the fact that TM is expressed in two distinct forms, Gerlitz et al. (9) postulated a model involving glycosyltransferase competition between xylosyltransferase and N-acetylgalactosaminyltransferase for Ser-474, whereas Lin et al. (10) suggested that occupation of the adjacent O-linked glycosylation may be important because of the steric hindrance for xylosyltransferase of the cell line.
If the new structures determined in the present paper link to a similar position, the following possibilities arise. The key step in producing the low molecular weight TM is the first GalNAc transfer reaction to the proteoglycan linkage tetrasaccharide regulated by sulfation at 3C of GlcA, which leads to regulation of the cell-surface anticoagulant potential. This is compatible with the finding that GalNAc transferase catalyzing chondroitin chain elongation cannot transfer GalNAc to 3-O-sulfated GlcA (30). This sulfation may be accomplished either with a similar sulfotransferase, as reported for the detection of SO 4 -3GlcA␤1-4Xyl␤-4-methylumbelliferone from the substrate, 4-methylumbelliferyl ␤-Xyl (31), or with the sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope (32,33). These results suggest that glycosaminoglycan chain elongation of human TM seems to be abolished at the linkage tetrasaccharide core structure by addition of a sulfate group, indicating that 3-O-sulfation is a stop signal leading to TM lacking chondroitin sulfate.