Identification and characterization of a novel UDP-GalNAc:GlcA[beta]-R [alpha]1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line

Hiroshi Kitagawa, Yuko Kano, Hiromi Shimakawa, Fumitaka Goto1, Tomoya Ogawa1,2, Hidetoshi Okabe3 and Kazuyuki Sugahara4

Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan, 1RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama, 351-01, Japan, 2Graduate School for Agriculture and Life Science, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan and 3Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Shiga, Japan

Received on October 22, 1998; revised on November 21, 1998; accepted on November 24, 1998

We recently discovered a novel [alpha]-N-acetylgalactosaminyltransferase in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) and also in mouse mastcytoma cells (Lidholt et al., Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an [alpha]-GalNAc residue to the linkage tetrasaccharide-serine, GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser, derived from proteoglycans. In this study, we characterized this enzyme using a preparation obtained from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl-Sepharose chromatography. Structural characterization by 1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser, as a substrate demonstrated that the enzyme was a UDP-GalNAc:GlcA[beta]1-R [alpha]1,4-N-acetylgalactosaminyltransferase. This is the first identification of an [alpha]1,4-N-acetylgalactosaminyltransferase. Using N-acetylchondrosine GlcA[beta]1-3GalNAc as an alternative substrate, the enzyme required divalent cations for the transferase reaction, with maximal activity at 20 mM Mn2+ and exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N-acetylchondrosine, the linkage tetrasaccharide-serine, and UDP-GalNAc were 1060 µM, 188 µM, and 27 µM, respectively. This suggested that the linkage tetrasaccharide-serine was a good acceptor substrate for the enzyme. In addition, the enzyme utilized glucuronylneolactotetraosylceramide GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer but not sulfoglucuronylneolactotetraosylceramide GlcA(3-O-sulfate)[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer as acceptor substrates. The possibility of involvement of this enzyme in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.

Key words: [alpha]1,4-N-acetylgalactosaminyltransferase/chondroitin sulfate/glycosaminoglycan/glycosyltransferase/proteoglycan

Introduction

Sulfated glycosaminoglycans including heparin/heparan sulfate, chondroitin sulfate, and dermatan sulfate are covalently bound to Ser residues in the core proteins through the common carbohydrate-protein linkage structure, GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser (for reviews, see Lindahl and Rodén, 1972; Rodén, 1980). Heparin/heparan sulfate is synthesized once GlcNAc is transferred to the common linkage region, whereas chondroitin sulfate is formed if GalNAc is added first. The two distinct transferases, which catalyze the transfer of GlcNAc or GalNAc, respectively, to the common linkage region, are the key enzymes which determine the glycosaminoglycan species to be synthesized. The transferases for the first GlcNAc and GalNAc residues are thought to be different from the N-acetylhexosaminyltransferases catalyzing the elongation steps, which transfer GlcNAc or GalNAc to the corresponding repeating disaccharide region (Lindahl and Rodén, 1972; Rodén, 1980). The molecular mechanisms are unknown for the synthesis of different glycosaminoglycans on the common linkage region.

While searching for the [beta]-N-acetylgalactosaminyltransferase that catalyzes the transfer of a [beta]-GalNAc to the common linkage region, we encountered a novel [alpha]-N-acetylgalactosaminyltransferase ([alpha]-GalNAcT) in fetal bovine serum and in mouse mastcytoma cells, which catalyzed the transfer of an [alpha]-GalNAc residue to the linkage tetrasaccharide-serine, GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser, derived from proteoglycans (Kitagawa et al., 1995; Lidholt et al., 1997). The positional assignment of the GalNAc attachment to the terminal GlcA had not been previously accomplished due to limited availability of authentic acceptor substrates and the requirement of a large amount of enzyme preparation. As we found substantial activity of [alpha]-GalNAcT in the culture medium of a human cancer cell line, we partially purified the enzyme in this study by phenyl-Sepharose chromatography and characterized the reaction product by 1H NMR spectroscopy analysis as well as the properties of the purified enzyme. The results demonstrated that the enzyme was a UDP-GalNAc:GlcA[beta]1-R [alpha]1,4-N-acetylgalactosaminyltransferase.

Results

Preparation of an [alpha]-GalNAcT solution from cell culture supernatant

The serum-free cell culture medium conditioned by human malignant fibrous histiocytoma cell line MFH-7 had [alpha]-GalNAcT activity of about 0.2 mIU/l and a total protein concentration of 0.167 g/l. Fractions from phenyl-Sepharose chromatography eluted by Buffer A showed high [alpha]-GalNAcT activities. These fractions were pooled and concentrated. The [alpha]-GalNAcT activity of the preparation was approximately 143 mIU/l and a total protein concentration of 39.1 g/l. Thus, the specific [alpha]-GalNAcT activity referring to the total protein content was increased 3-fold from 1.2 mIU/g to 3.7 mIU/g. Further studies were performed with this preparation ([alpha]-GalNAcT).

Table I. 1H chemical shifts of structural-reporter-groups of the constituent monosaccharides of the [alpha]-GalNAcT reaction product
Residue   Product (GalNAc-GlcA-Gal-Gal-Xyl-Ser) R1
Ser [alpha] 3.943 3.94
[beta] 3.694 3.69
Xyl-1 H-1 4.450 4.44
H-2 3.363 3.35
H-3 3.611 3.61
H-4 3.86 a 3.87
H-5ax 3.404 3.41
H-5eq 4.114 4.12
Gal-2 H-1 4.531 4.54
H-2 3.68 3.68
H-3 3.83 3.83
H-4 4.188 4.19
H-5 3.75 3.70-3.80
H-6 3.70-3.80 3.70-3.80
Gal-3 H-1 4.663 4.67
H-2 3.73 3.74
H-3 3.78 3.79
H-4 4.156 4.16
H-5 3.70-3.80 3.70-3.80
H-6 3.70-3.80 3.70-3.80
GlcA-4 H-1 4.658 4.66
H-2 3.424 3.43
H-3 3.68 3.68
H-4 3.71 3.70-3.75
H-5 3.72 3.70-3.75
GalNAc-5 H-1 5.441 5.45
H-2 4.160 4.17
H-3 3.87 3.88
H-4 3.97 3.99
H-5 3.70-3.80 3.70-3.80
H-6 3.70-3.80 3.70-3.80
NAc 2.050 2.05
1H chemical shifts of the constituent monosaccharides of the [alpha]-GalNAcT reaction product are shown together with those of the synthetic reference pentasaccharide-serine; R1, GalNAc[alpha]1-4GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser (Tamura et al., 1996). Chemical shifts are given in p.p.m. downfield from internal sodium 4, 4-dimethyl-4-silapentane-1-sulfonate (Vliegenthart et al., 1983) but were actually measured indirectly to acetone in D2O ([delta] 2.225) at 26°C.
aThe estimated error for the values to two decimal places is only ±0.01 p.p.m. due to partial overlap of signals. That for the values to three decimal places is ±0.001 p.p.m..

Structural characterization of the reaction product by 500 MHz 1H NMR spectroscopy

As the positional assignment of GalNAc attachment to the terminal GlcA had not been previously accomplished, we first attempted to characterize the reaction product by 1H NMR spectroscopy. For this analysis, the reaction mixture obtained by incubation of the [alpha]-GalNAcT preparation with the linkage tetrasaccharide-serine GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser was subjected to gel filtration on a column of Superdex 30. As shown in Figure 1, the reaction product eluted at the position corresponding to the pentasaccharide-serine was pooled, desalted by repeated evaporation, reconstituted in D2O and analyzed by 1H NMR spectroscopy. The one-dimensional spectrum of the isolated product was recorded at 26°C and is shown in Figure 2. Signals at [delta] 4.4-5.5 p.p.m. were identified as H-1 resonances of the constituent saccharide residues by comparison with the NMR spectra of the synthetic reference compound GalNAc[alpha]1-4GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser (Tamura et al., 1996). Proton signals in the one-dimensional spectrum were assigned as described previously (Sugahara et al., 1988, 1991, 1992; de Waard et al., 1992) using the two-dimensional COSY spectrum (data not shown). The NMR data of the product are summarized in Table I together with those of the synthetic reference compound (Tamura et al., 1996). These results indicated that the observed chemical shifts of the product were virtually identical to those obtained for the synthetic reference compound GalNAc[alpha]1-4GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser (Tamura et al., 1996). Based on these data, we concluded that the [alpha]-GalNAcT was capable of catalyzing the transfer of a GalNAc residue from UDP-GalNAc through an [alpha]1,4 linkage to terminal [beta]-linked GlcA residues on acceptor molecules and thus can be defined as a UDP-GalNAc:GlcA[beta]-R [alpha]1,4-N-acetylgalactosaminyltransferase ([alpha]4-GalNAcT).


Figure 1. Gel filtration analysis of [alpha]-GalNAcT reaction product obtained with the tetrasaccharide-serine as an acceptor. The [alpha]-GalNAcT reaction was carried out in the presence (solid line) or absence (dashed line) of the linkage tetrasaccharide-serine GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser as an acceptor as described in Materials and methods. The reaction mixtures were subjected to gel filtration on a column of Superdex 30 (1.6 × 60 cm) equilibrated with 0.25 M NH4HCO3/7% 1-propanol. Fractions (1 ml each) were collected at a flow rate of 1.0 ml/min and analyzed for radioactivity. The radioactive peak corresponding to the [alpha]-GalNAcT reaction product, the putative linkage pentasaccharide-serine (indicated by the bar) was pooled and desalted by repeated evaporation.


Figure 2. Structural-reporter-group regions of the 500 MHz 1H NMR spectrum of the [alpha]-GalNAcT reaction product recorded in D2O at 26°C. One-dimensional spectrum of the [alpha]-GalNAcT reaction product. The letters and numbers refer to the corresponding residues in the structure.

Properties of [alpha]4-GalNAcT

When the linkage tetrasaccharide-serine was used for the [alpha]4-GalNAcT assay, the major problem appeared to be the limitation of time-consuming and laborious conventional gel filtration for separation of the products from the unincorporated UDP-[3H]GalNAc (see Figure 1). Therefore, we developed a rapid and simple assay method using N-acetylchondrosine GlcA[beta]1-3GalNAc as a substrate (see Materials and methods) as [alpha]4-GalNAcT utilizes not only the linkage tetrasaccharide-serine but various regular oligosaccharides with a GlcA residue at the nonreducing terminus as acceptor substrates (Kitagawa et al., 1995, 1997a). Using this method, the properties of [alpha]4-GalNAcT were determined as below.

Figure 3 shows the effects of buffers and pHs on the partially purified [alpha]4-GalNAcT. The enzyme exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM MES buffer at pH 6.5 (Figure 3). Divalent cations were essential for the enzymatic reaction and 10 mM EDTA completely abolished the activity (Figure 4A). Mn2+ exhibited the highest activity under standard assay conditions, and Co2+ was 40% as effective as Mn2+ (Figure 4A). The optimal concentration of Mn2+ was ~20 mM (Figure 4B). Under the established standard incubation conditions described in Materials and methods, GalNAc incorporation into N-acetylchondrosine was proportional to the incubation time for up to 8 h (Figure 5).


Figure 3. Effects of buffers and pHs on the activity of the [alpha]4-GalNAcT. The effects of pH on the GalNAc transfer to N-acetylchondrosine GlcA[beta]1-3GalNAc were determined under standard assay conditions with different buffers at a final concentration of 50 mM. The buffers are sodium acetate (solid triangles), sodium cacodylate (open squares), MES-NaOH (solid circles), imidazole-HCl (open circles), and Tris-HCl (open triangles). Assays proceeded as described in Materials and methods. Data represents one of two series of independent experiments, where the two series of experiments gave essentially identical results.


Figure 4. Effects of divalent cations (A) and Mn2+ concentration (B) on the [alpha]4-GalNAcT activity. (A) The effects of divalent cations on the GalNAc transfer to N-acetylchondrosine were determined under standard assay conditions with different divalent cations or EDTA at a final concentration of 10 mM. (B) The effects of Mn2+ concentrations on the GalNAc transfer to N-acetylchondrosine were determined under standard assay conditions, except that the concentration of MnCl2 was varied. Assays proceeded as described in Materials and methods. Data represents one of two series of independent experiments, where the two series of experiments gave essentially identical results.


Figure 5. Dependence of the [alpha]4-GalNAcT activity on the incubation time. The rate of GalNAc transfer to N-acetylchondrosine as a function of the incubation time was determined as described in Materials and methods. Data represents one of two series of independent experiments, where the two series of experiments gave essentially identical results.

To investigate the effects of concentrations of the acceptor N-acetylchondrosine and the donor UDP-GalNAc, we determined some of the kinetic parameters of the [alpha]4-GalNAcT. As shown in Figure 6A,B, the apparent Km values for N-acetylchondrosine and UDP-GalNAc were 1060 µM and 27 µM, respectively. To further investigate the possible involvement of the [alpha]4-GalNAcT in glycosaminoglycan biosynthesis, the apparent Km value for the linkage tetrasaccharide-serine was also determined. As shown in Figure 6C, it was 188 µM.


Figure 6. Effects of concentrations of N-acetylchondrosine, UDP-GalNAc, and the linkage tetrasaccharide-serine on the [alpha]4-GalNAcT activity. N-Acetylchondrosine (A), UDP-GalNAc (B) or the linkage tetrasaccharide-serine GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser (C) was added to the established assay mixture at different final concentrations, and assays proceeded as described in Materials and methods. Data represents one of two series of independent experiments, where the two series of experiments gave essentially identical results.

Since no [alpha]-GalNAc-capped structure has been reported in naturally occurring glycosaminoglycan chains, it is possible that the [alpha]4-GalNAcT participates in the biosynthesis of other classes of GlcA-containing glycoconjugates, e.g., GlcA[beta]1-3Gal[beta]1-4GlcNAc-R, precursor of the HNK-1 carbohydrate epitope on glycoproteins or glycolipids. Thus, glucuronylneolactotetraosylceramide, GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer and sulfoglucuronylneolactotetraosylceramide, GlcA(3-O-sulfate)[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer were tested as acceptor using the [alpha]4-GalNAcT preparation. As shown in Table II, only the former compound served as an acceptor substrate although the linkage tetrasaccharide-serine was a better substrate than that.

Discussion

In this study, we characterized a unique [alpha]-N-acetylgalactosaminyltransferase which catalyzes the transfer of an [alpha]-GalNAc to the linkage tetrasaccharide- and hexasaccharide-serines derived from chondroitin sulfate proteoglycans and to the even-numbered regular chondro-oligosaccharides with a GlcA residue at the nonreducing terminus as acceptor substrates (Kitagawa et al., 1995). Structural characterization by 1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser, as a substrate clearly demonstrated that the enzyme was a UDP-GalNAc:GlcA[beta]1-R [alpha]1,4-N-acetylgalactosaminyltransferase. To our knowledge, three other types of [alpha]-GalNAc transferases have so far been characterized in detail. One type is a family of the polypeptide [alpha]-GalNAc transferases responsible for the biosynthetic initiation of O-linked oligosaccharides, which catalyzes the transfer of an [alpha]-GalNAc residue to a serine or a threonine residue on the protein acceptor (Clausen and Bennett, 1996). The other two are [alpha]1,3-N-acetylgalactosaminyltransferases, which are involved in the synthesis of the Forssman antigen GalNAc[alpha]1-3GalNAc[beta]1-3Gal[alpha]1-4Gal[beta]1-4Glc (Taniguchi et al., 1982) and the blood group A-specific antigen GalNAc[alpha]1-3(Fuc[alpha]1-2)Gal[beta]1-3GlcNAc[beta]-R (Clausen et al., 1990), respectively. We clearly revealed that [alpha]4-GalNAcT is distinct from these three types of [alpha]-GalNAc transferases. Thus, this is the first identification of an [alpha]1,4-N-acetylgalactosaminyltransferase.

Table II. Acceptor specificity of the [alpha]4-GalNAcT
Acceptor Relative rate (%)
GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser 100
GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer 22
GlcA(3-O-sulfate)[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer 0
Various acceptor substrates (1 nmol each) were incubated with the [alpha]4-GalNAcT as described in Materials and methods. Relative incorporation rates were calculated as percentages of the incorporation obtained with the linkage tetrasaccharide-serine GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser. The data represent one of two series of independent experiments, where the two series of experiments gave essentially identical results.

Recently, Etchison et al. (1995) reported that when several different cell lines in culture were labeled with [3H]galactose in the presence of 4-methyl umbelliferyl [beta]-d-xyloside (Xyl[beta]4MU), a small portion of the labeled products contained the carbohydrate-protein linkage region of chondroitin sulfate, which is, however, terminating with an [alpha]-GalNAc residue instead of a typical [beta]-GalNAc residue. Moreover, structural analysis of the labeled product by 1H NMR spectroscopy revealed that the GalNAc was linked to the nonreducing terminal GlcA residue through an [alpha]1-4 linkage (Manzi et al., 1995). In view of the fact that the [alpha]4-GalNAcT identified in the present study was capable of catalyzing the transfer of a GalNAc residue from UDP-GalNAc through an [alpha]1,4 linkage to a terminal [beta]-linked GlcA residue on the linkage tetrasaccharide-serine and that the apparent Km value for the linkage tetrasaccharide-serine was 188 µM (see Figure 6C), it was most likely that this was the [alpha]4-GalNAcT that catalyzed the formation of the product terminating with an [alpha]1,4-linked GalNAc unit in the cultured cells.

As no [alpha]-GalNAc-capped structure has been reported in naturally occurring glycosaminoglycan chains, the possible role of the [alpha]4-GalNAcT in glycosaminoglycan biosynthesis remains unclear. We recently revealed that the [alpha]-GalNAc-capped pentasaccharide serine GalNAc[alpha]1-4GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser, a reaction product of the [alpha]4-GalNAcT, was not utilized as an acceptor for a glucuronyltransferase involved in chondroitin sulfate biosynthesis (Kitagawa et al., 1997b). Therefore, we anticipated that the addition of an [alpha]-GalNAc unit to the tetrasaccharide core of the linkage region of proteoglycans might serve as a stop signal that precludes further chain elongation, creating a part-time proteoglycan (Fransson, 1987). To search for the [alpha]-GalNAc-capped pentasaccharide linkage structure GalNAc[alpha]1-4GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl on part-time proteoglycans, we isolated and characterized the O-linked oligosaccharides on recombinant human [alpha]-thrombomodulin, a part-time proteoglycan (Nadanaka et al., 1998). Structural analysis unexpectedly revealed the occurrence of an immature truncated sequence GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl on the [alpha]-thrombomodulin (Nadanaka et al., 1998). Although further study of the carbohydrate structure on other part-time proteoglycans is necessary to confirm the generality of the finding, it is unlikely that the [alpha]4-GalNAcT is involved in the formation of a part-time proteoglycan.

On the other hand, as the apparent Km values of the [alpha]4-GalNAcT for GlcA-containing oligosaccharides were low as revealed in this study and a substantial amount of [alpha]4-GalNAcT activity was detected in Golgi fractions prepared from several cells (Lidholt et al., 1997; Miura and Freeze, 1998), it is possible that the [alpha]4-GalNAcT participates in the biosynthesis of other classes of GlcA-containing glycoconjugates, e.g., N-linked glycoproteins and glycolipids. Indeed, considering the similarity in the terminal structures of the linkage tetrasaccharide GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl and precursor of the HNK-1 carbohydrate epitope on glycoproteins or glycolipids, GlcA[beta]1-3Gal[beta]1-4GlcNAc-R, it is reasonable to assume that the [alpha]4-GalNAcT utilizes these classes of glycoconjugates as acceptor substrates. In this regard, it should be noted that when glucuronylneolactotetraosylceramide, GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer and sulfoglucuronylneolactotetraosylceramide, GlcA(3-O-sulfate)[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer were tested as acceptor using the [alpha]4-GalNAcT preparation, only the former compound served as an acceptor substrate (see Table II). In addition, Miura et al. (1998) reported that GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]-octyl also served as an acceptor substrate for the [alpha]4-GalNAcT and that the apparent Km value for this substrate was 0.5 mM. Although these [alpha]-GalNAc-capped structures have not been reported in naturally occurring glycoproteins or glycolipids, the [alpha]4-GalNAcT might play an important role in the regulation of the expression of the HNK-1 carbohydrate epitope. Identification of [alpha]-GalNAc-capped structures in naturally occurring glycoconjugates and purification and molecular cloning of the [alpha]4-GalNAcT may reveal supportive evidence.

Materials and methods

Materials

UDP-[3H]GalNAc (6.3 Ci/mmol) and unlabeled UDP-GalNAc were purchased from New England Nuclear and Sigma, respectively. The linkage tetrasaccharide-serine GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl[beta]1-O-Ser was chemically synthesized (Goto and Ogawa, 1993). N-Acetylchondrosine GlcA[beta]1-3GalNAc was provided by Dr. K.Yoshida (Seikagaku Corp.). Glucuronylneolactotetraosylceramide, GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer and sulfoglucuronylneolactotetraosylceramide, GlcA(3-O-sulfate)[beta]1-3Gal[beta]1-4GlcNAc[beta]1-3Gal[beta]1-4Glc[beta]1-1Cer were obtained from Wako, Osaka, Japan. HiLoad 16/60 Superdex 30 (prep grade) and phenyl-Sepharose were obtained from Amersham Pharmacia Biotech, Uppsala, Sweden. All other reagents and chemicals were of the highest quality.

Preparation of an [alpha]-GalNAcT-enriched solution from human malignant fibrous histiocytoma cell (MFH-7) cultures

MFH-7 cells (established by Dr. Hidetoshi Okabe, Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Shiga, Japan) were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum. After the cells reached confluence, they were incubated in serum-free medium Media I (IBL, Fujioka, Japan) for 3 days. Thereafter, the culture medium was replaced every 3 days with fresh Media I and the cell culture in Media I was continued for 15 days. The spent Media I was pooled and centrifuged at 10,000 × g for 10 min to remove cell debris. To the supernatant solution, (NH4)2SO4 and glycerol were added to the final concentrations 1.0 M and 10% (v/v), respectively. The medium fraction (1 l) was applied to a column of phenyl-Sepharose (15.0 ml) equilibrated with 10% glycerol in 10 mM MES-NaOH, pH 6.5 (Buffer A) containing 1.0 M (NH4)2SO4, washed with 150 ml of Buffer A containing 0.6 M (NH4)2SO4, and then eluted with 45 ml of Buffer A. Chromatography was repeated twice. The eluates from the two chromatographies were combined, concentrated, and then dialyzed against 15 ml of Buffer A containing 0.15 M NaCl twice using a Centriplus-50 concentrator (Amicon Inc.).

[alpha]-GalNAc transferase assay

As shown in Results, the assay conditions for [alpha]-GalNAcT were established by examining various factors such as buffers, metal ions, substrate concentrations and inhibitors for UDP-GalNAc degradation. Unless otherwise noted, the standard assay mixture contained 2 µl of an enzyme solution corresponding to 20 nIU of the enzyme, 5 nmol of N-acetylchondrosine GlcA[beta]1-3GalNAc, 8.57 µM UDP-[3H]GalNAc (5.28 × 105 d.p.m.), 20 mM MnCl2, 171 µM ATP in a total volume of 35 µl of 50 mM MES buffer, pH 6.5. ATP was included to prevent enzymatic degradation of UDP-GalNAc. The reaction mixtures were incubated at 37°C for 4 h and then diluted in 1 ml 5 mM sodium phosphate, pH 6.8. 3H-Labeled products were separated from UDP-[3H]GalNAc by passage of the reaction mixture through a Pasteur pipette column containing Dowex 1-X8 (PO42-, 100-400 mesh; Bio-Rad) as described previously (Kitagawa et al., 1997a). The isolated products were quantified in a scintillation spectrophotometer. Net [3H]GalNAc incorporation into the N-acetylchondrosine was calculated by subtracting the blank value obtained in its absence. Under the incubation conditions for [alpha]-GalNAcT, GalNAc incorporation into N-acetylchondrosine was proportional to the incubation period for up to 8 h (see Figure 5).

Preparation of the enzyme reaction product

Large-scale incubation with the linkage tetrasaccharide-serine as an acceptor was performed to isolate a sufficient product for characterization by 500 MHz 1H NMR spectroscopy. Incubation mixtures contained the following constituents in a total volume of 625 µl of 100 mM MES buffer, pH 6.5: 25 nmol of the linkage tetrasaccharide-serine, 200 µM UDP-[3H]GalNAc (5.04 × 106 d.p.m.), 20 mM MnCl2, 1 mM ATP, and 175 µl of the enzyme solution corresponding to 25 µIU of the enzyme. Reaction mixtures were incubated at 37°C for 20 h and the reactions were terminated by immersing the reaction tubes in boiling water for 1 min. The mixtures were subjected to gel filtration on a column of Superdex 30 (1.6 × 60 cm) equilibrated with 0.25 M NH4HCO3/7% 1-propanol. Fractions (1 ml each) were collected at a rate of 1.0 ml/min and analyzed for radioactivity. The radioactive peak corresponding to the [alpha]-GalNAcT reaction product, the putative linkage pentasaccharide-serine, (indicated by the bar in Figure 1) was pooled, evaporated to dryness for desalting, and then reconstituted in water.

500 MHz 1H NMR spectroscopy

The isolated reaction product (ca. 12 nmol) was repeatedly exchanged in D2O with intermediate lyophilization. The 1H NMR spectra was measured in a Varian VXR-500 using a nanoprobe at 26°C. Chemical shifts were given relative to sodium 4,4-dimethyl-4-silapentane-1-sulfonate but were actually measured indirectly, relative to acetone ([delta] 2.225) in D2O (Vliegenthart et al., 1983).

Acknowledgments

This work was supported in part by the Science Research Promotion Fund of the Japan Private School Promotion Foundation (to K.S.), the Research Grant from the Research Foundation for Parmaceutical Sciences (to H.K.) and a Grant-in-aid for Encouragement of Young Scientists 09772013 (to H.K.), a Grant-in-aid for Scientific Research (B) 09470509 (to K.S.), and a Grant-in-aid for Scientific Research on Priority Areas 10178102 (to K.S.) from the Ministry of Education, Science, Culture, and Sports of Japan.

Abbreviations

GalNAc, N-acetyl-d-galactosamine; GlcA, d-glucuronic acid; MES, 2-(N-morpholino)ethanesulfonic acid; COSY, correlation spectroscopy.

References

Clausen ,H. and Bennett,E.P. (1996) A family of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferases control the initiation of mucin-type O-linked glycosylation. Glycobiology, 6, 635-646. MEDLINE Abstract

Clausen ,H., White,T., Takio,K., Titani,K., Stroud,M., Holmes,E., Karkov,J., Thim,L. and Hakomori,S. (1990) Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc[alpha]1-2Gal [alpha]1-3-N-acetylgalactosaminyltransferase from human lung tissue. J. Biol. Chem., 265, 1139-1145. MEDLINE Abstract

de Waard ,P., Vliegenthart,J.F.G., Harada,T. and Sugahara,K. (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues. J. Biol. Chem., 267, 6036-6043. MEDLINE Abstract

Etchison ,J.R., Srikrishna,G. and Freeze,H.H. (1995) A novel method to co-localize glycosamonoglycan-core oligosaccharide glycosyltransferases in rat liver Golgi: co-localization of galactosyltransferase I with a sialyltransferase. J. Biol. Chem., 270, 756-764. MEDLINE Abstract

Fransson ,L.Å. (1987) Structure and function of cell-associated proteoglycans. Trends Biochem. Sci., 12, 406-411.

Goto ,F. and Ogawa,T. (1993) Recent aspects of glycoconjugate synthesis. A synthetic approach to the linkage region of proteoglycans. Pure Appl. Chem., 65, 793-801.

Kitagawa ,H., Tanaka,Y., Tsuchida,K., Goto,F., Ogawa,T., Lidholt,K., Lindahl,U. and Sugahara,K. (1995) N-Acetylgalactosamine (GalNAc) transfer to the common carbohydrate-protein linkage region of sulfated glycosaminoglycans: identification of UDP-GalNAc:chondro-oligosaccharide [alpha]-N-acetylgalactosaminyltransferase in fetal bovine serum. J. Biol. Chem., 270, 22190-22195. MEDLINE Abstract

Kitagawa ,H., Tsutsumi,K., Ujikawa,M., Goto,F., Tamura,J., Neumann,K.W., Ogawa,T. and Sugahara,K. (1997a) Regulation of chondroitin sulfate biosynthesis by specific sulfation: acceptor specificity of serum [beta]-GalNAc transferase revealed by structurally-defined oligosaccharides. Glycobiology, 7, 531-537. MEDLINE Abstract

Kitagawa ,H., Ujikawa,M., Tsutsumi,K., Tamura,J., Neumann,K.W., Ogawa,T. and Sugahara,K. (1997b) Characterization of serum [beta]-glucuronyltransferase involved in chondroitin sulfate biosynthesis. Glycobiology, 7, 905-911. MEDLINE Abstract

Lidholt ,K., Fjelstad,M., Lindahl,U., Goto,F., Ogawa,T., Kitagawa,H. and Sugahara, K. (1997) Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma. Glycoconjugate J., 14, 737-742.

Lindahl ,U. and Rodén,L. (1972) Carbohydrate-peptide linkages in proteoglycans of animal, plant and bacterial origin. In Gottschalk,A. (ed.), Glycoproteins. Elsevier, New York, pp. 491-517.

Manzi ,A., Salimath,P.V., Spiro,R.C., Keifer,P.A. and Freeze,H.H. (1995) Identification of a novel glycosaminoglycan core-like molecule I: 500 MHz 1H NMR analysis using a nano-NMR probe indicates the presence of a terminal [alpha]-GalNAc residue capping 4-methylumbelliferyl-[beta]-d-xyloside. J. Biol. Chem., 270, 9154-9163. MEDLINE Abstract

Miura ,Y. and Freeze,H.H. (1998) [alpha]-N-Acetylgalactosamine-capping of chondroitin sulfate core region oligosaccharides primed on xylosides. Glycobiology, 8, 813-819. MEDLINE Abstract

Miura ,Y., Manzi,A., Ding,Y., Hindsgaul,O. and Freeze,H.H. (1998) [alpha]-N-Acetylgalactosamine-capping of glucuronides in animal cells. XIX International Carbohydrate Symposium (abstracts). August 9-14, 1998, San Diego, CA, abstract no. CP070.

Nadanaka ,S., Kitagawa,H. and Sugahara,K. (1998) Demonstration of the immature glycosaminoglycan tetrasaccharide sequence GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl on recombinant soluble human [alpha]-thrombomodulin: an oligosaccharide structure on a 'part-time" proteoglycan. J. Biol. Chem., 273, 33728-33734. MEDLINE Abstract

Rodén ,L. (1980) Structure and metabolism of connective tissue proteoglycans. In Lennarz,W.J. (ed.), The Biochemistry of Glycoproteins and Proteoglycans. Plenum, New York, pp. 267-371.

Sugahara ,K., Yamashina,I., de Waard,P., van Halbeek,H. and Vliegenthart,J.F.G. (1988) Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans of Swarm rat chondrosarcoma. J. Biol. Chem., 263, 10168-10174. MEDLINE Abstract

Sugahara ,K., Masuda,M., Harada,T., Yamashina,I., de Waard,P. and Vliegenthart,J.F.G. (1991) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage. Eur. J. Biochem., 202, 805-811. MEDLINE Abstract

Sugahara ,K., Ohi,Y., Harada,T., de Waard,P. and Vliegenthart,J.F.G. (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and/or phosphate residue. J. Biol. Chem., 267, 6027-6035. MEDLINE Abstract

Tamura ,J., Neumann,K.W. and Ogawa,T. (1996) Synthetic studies of glycosyl serines in the carbohydrate-protein region of proteoglycans. Liebigs Ann., 1239-1257.

Taniguchi ,N., Yokosawa,N., Gasa,S. and Makita,A. (1982) UDP-N-Acetylgalactosamine:globoside [alpha]-3-N-acetylgalactosaminyltransferase: purification, characterization, and some properties. J. Biol. Chem., 257, 10631-10637. MEDLINE Abstract

Vliegenthart ,J.F.G., Dorland,L. and van Halbeek,H. (1983) High-resolution 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohyr. Chem. Biochem., 41, 209-374.


4To whom correspondence should be addressed at: Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan


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