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.
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.
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 |
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
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
Figure
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
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.
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 | 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 |
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
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
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
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).
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.
GalNAc, N-acetyl-d-galactosamine; GlcA, d-glucuronic acid; MES, 2-(N-morpholino)ethanesulfonic acid; COSY, correlation spectroscopy.
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 10 Jun 1999
Copyright©Oxford University Press, 1999.