Characterization of mammalian UDP-GalNAc:glucuronide [alpha]1-4-N-acetylgalactosaminyltransferase

Yoshiaki Miura, Yili Ding, Adriana Manzi1, Ole Hindsgaul and Hudson H.Freeze2

The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA and 1Nextran, Inc., San Diego, CA 92121, USA

Received on February 5, 1999; revised on March 18, 1999; accepted on March 26, 1999

We previously reported that cultured cells incubated with [beta]-xylosides synthesized [alpha]-GalNAc-capped GAG-related xylosides, GalNAc[alpha]GlcA[beta]Gal[beta]Gal[beta]Xyl[beta]-R and GalNAc[alpha]GlcA[beta]GalNAc[beta]GlcA[beta]Gal[beta]Gal[beta]Xyl[beta]-R, where R is 4-methylumbelliferyl or p-nitrophenyl (Manzi et al., 1995; Miura and Freeze, 1998). In this study, we characterized an [alpha]-N-acetylgalactosaminyltransferase ([alpha]-GalNAc-T) that probably adds the [alpha]-GalNAc residue to the above xylosides. Microsomes from several animal cells and mouse brain contained the enzyme activity which requires divalent cations, and has a relatively broad pH optimal range around neutral. The apparent Km values were in the submillimolar range for the acceptors tested, and 19 µM for UDP-GalNAc. 1H-NMR analysis of the GlcA-[beta]-MU acceptor product showed the GalNAc residue is transferred in [alpha]1,4-linkage to the glucuronide, which is consistent with previous results reported on [alpha]-GalNAc-capped Xyl-MU (Manzi et al., 1995). Various artificial glucuronides were tested as acceptors to assess the influence of the aglycone. Glucuronides with a bicyclic aromatic ring, such as 4-methylumbelliferyl [beta]-D-glucuronide (GlcA-[beta]-MU) and [alpha]-naphthyl [beta]-D-glucuronide, were the best acceptors. Interestingly, a synthetic acceptor that resembles the HNK-1 carbohydrate epitope but lacking the sulfate group, GlcA[beta]1,3Gal[beta]1,4GlcNAc[beta]-O-octyl ([Delta]SHNK-C8), was a better acceptor for [alpha]-GalNAc-T than the glycosaminoglycan-protein linkage region tetrasaccharyl xyloside, GlcA[beta]1,3Gal[beta]1,3Gal[beta]1,4Xyl[beta]-MU. GlcA-[beta]-MU and [Delta]SHNK-C8 competed for the [alpha]-GalNAc-T activity, suggesting that the same activity catalyzes the transfer of the GalNAc residue to both acceptors. Taken together, the results show that the [alpha]-GalNAc-T described here is not restricted to GAG-type oligosaccharide acceptors, but rather is a UDP-GalNAc:glucuronide [alpha]1-4-N-acetylgalactosaminyltransferase.

Key words: [alpha]-N-acetylgalactosaminyltransferase/ chondroitin sulfate/[beta]-glucuronides/HNK-1 epitope/glycosaminoglycan

Introduction

We previously reported the formation of [alpha]-GalNAc-terminated xylosides by several types of cells. The terminal GalNAc residue is in an [alpha]1,4-linkage to glucuronic acid (GlcA) of glycosaminoglycan-protein linkage region tetrasaccharyl xylosides secreted into culture medium during the incubation of the cells with xyloside primers, such as 4-methylumbelliferyl [beta]-D-xyloside (Xyl-[beta]-MU) and p-nitrophenyl [beta]-D-xyloside (Xyl-[beta]-pNP) (Manzi et al., 1995; Salimath et al., 1995). Recently, we have also shown that similar alpha-GalNAc-capping was formed on a nonreducing terminal GlcA of chondroitin sulfate type repeating unit built on xylosides, GlcA[beta]GalNAc[beta]GlcA[beta]Gal[beta]Gal[beta]Xyl-[beta]-MU (Miura and Freeze, 1998). An enzyme activity capable of transferring [alpha]-GalNAc to chondro-oligosaccharides has been detected in fetal bovine serum by Sugahara and his colleagues (Kitagawa et al., 1995a). Despite efforts to identify naturally occurring glycoconjugates which possess a terminal GalNAc-[alpha]1,4-GlcA-[beta]-R sequence, none has been found so far. To define the range of possible target glycoconjugates, we studied the [alpha]-N-acetylgalactosaminyltransferase activity toward various molecules containing nonreducing terminal [beta]-GlcA residues using the enzyme present in microsomes from various cultured cell lines and mouse brain homogenates. The enzyme transferred GalNAc from UDP-GalNAc to a nonreducing [beta]-GlcA residue of several synthetic glucuronides, to the linkage region tetrasaccharyl xyloside, GlcA[beta]1,3Gal[beta]1,3Gal[beta]1,4Xyl[beta]-MU, and to a synthetic oligosaccharide, GlcA[beta]1,3Gal[beta]1, 4GlcNAc[beta]-O-octyl ([Delta]SHNK-C8). Among the acceptors tested, the glucuronides with a bicyclic aromatic ring and [Delta]SHNK-C8 were the best. Thus, based on the substrate specificity, we designate the enzymatic activity as UDP-GalNAc:glucuronide [alpha]1-4-N-acetylgalactosaminyltransferase to indicate that it is not restricted to chondro-oligosaccharide acceptors.

Results

GalNAc transferase activity in cells and mouse brain microsomes

We previously showed that microsomal fractions from human melanoma cells M21 contain substantial amounts of an [alpha]-N-acetylgalactosaminyltransferase ([alpha]-GalNAc-T) activity which transfers GalNAc from UDP-GalNAc to 4-methylumbelliferyl [beta]-D-glucuronide (GlcA-[beta]-MU) (Miura and Freeze, 1998). To examine the expression of this enzyme in animal cells, microsomes prepared from several cultured cell lines and fetal mouse brain were used as an enzyme source. Figure 1 shows that [alpha]-GalNAc-T activity was abundant in microsomal fractions of several cells and fetal mouse brain at day 19 (FMB19). Among those microsomes tested, Chinese hamster ovary (CHO) cells and FMB19 have the highest activity, while virtually no activity was detected in human fibroblasts and very little in hepatoma C3A cells, a derivative of HepG2 cells. Adult mouse and rat brain microsomal fractions also contained 30-80% of the activity of FMB19 (data not shown).


Figure 1. [alpha]-GalNAc-T activity in microsomal fractions. Assays were done using UDP-[3H]GalNAc as donor and 0.5 mM of GlcA-[beta]-MU as acceptor in standard conditions as described in Materials and methods. The results represent one of two series of experiments.

Properties of the [alpha]-GalNAc-T activity in fetal mouse brain

We chose fetal mouse brain microsomes to assess the enzyme properties because of its abundance, availability, and stability. Using GlcA-[beta]-MU as an acceptor, the activity showed a broad pH profile with optimum activity at pH 7 (Figure 2A). The enzyme activity required divalent cations, primarily Mn2+, and was abolished with 10 mM EDTA, as shown in Figure 2B. The activity was maximal in the presence of both Mn2+ and Mg2+, which were used in subsequent standard assays. The microsomes prepared as described in Materials and methods retained full [alpha]-GalNAc-T activity for over 6 months when stored at -80°C without repetitive freeze and thaw. Addition of dithiothreitol at 20 mM had no effect on enzyme activity (data not shown). Under the standardized conditions described in Materials and methods, the amount of GalNAc transfer to GlcA-[beta]-MU was proportional to the incubation time and the quantity of microsomal protein up to 4 h and 3 mg/ml, respectively (data not shown).


Figure 2. Effects of pH (A) and divalent cations (B) on the [alpha]-GalNAc-T activity present in FMB19 microsomal fraction. (A) The pH dependency of [alpha]-GalNAc-T activity was measured using MES buffer (open circles) and HEPES buffer (solid squares) at a final concentration of 50 mM. (B) The assays were done under standard conditions with different divalent cation chlorides or EDTA at a final concentrations of 10 mM. (N.D., not detected).

Structural analysis of the product obtained from GlcA-[beta]-MU

We reported previously that the [alpha]-GalNAc-capped xyloside produced by human melanoma cells has an [alpha]1,4-linked GalNAc to GlcA in the pentasaccharyl xyloside (Manzi et al., 1995). To confirm that the same activity catalyzes the transfer of GalNAc to GlcA-[beta]-MU in the fetal mouse brain microsomal fraction, we analyzed the structure of the product. Based on glycosidase susceptibilities of the product toward chicken liver [alpha]-N-acetylgalactosaminidase and complete resistance to [beta]-hexosaminidase and [beta]-glucuronidase, it is obvious that the GalNAc residue was transferred in [alpha]- rather than [beta]-linkage to glucuronides (data not shown). Using a large-scale reaction with GlcA-[beta]-MU as acceptor, the product was purified using a C18 cartridge, silica gel cartridge, and amine adsorption HPLC chromatography.

The recovered compound was analyzed by one-dimensional and two-dimensional 1H-NMR spectroscopy. The reference compound, GlcA-[beta]-MU, was analyzed in the same fashion for comparison. The one-dimensional 1H-NMR spectrum obtained with ~20 µg of the product, GalNAc[alpha]-GlcA-[beta]-MU, using a Nano-NMR probe is shown in Figure 3A. As expected, only two anomeric protons were observed, a [beta]-anomeric signal of GlcA at [delta] 5.185 (J1,2 = 8.5) and an [alpha]-anomeric signal of GalNAc at [delta] 5.430 (J1,2 = 3.5). Figure 3 also shows the homonuclear, two-dimensional TOCSY (Figure 3B) and DQFCOSY (Figure 3C) spectra. Assignments were made based on the 2D analysis and compared with those we previously reported (Manzi et al., 1995). The chemical shifts in D2O solution of the two monosaccharide units correspond well with those of GalNAc[alpha](1-4)GlcA[beta](1-3)Gal[beta](1-3)Gal[beta](1-4)Xyl-MU (Manzi et al., 1995). In order to confirm the position of linkage of the GalNAc unit, a two-dimensional ROESY experiment was carried out. The only ROE cross peak observed between the residues was between the anomeric signal of [alpha]-GalNAc and H-4 of GlcA (data not shown).


Figure 3. NMR spectra of GalNAc[alpha]-GlcA-[beta]-MU recorded at 500 MHz in D2O at 30°C. (A) One-dimensional complete 1H-NMR spectrum of GalNAc[alpha]-GlcA-[beta]-MU. (B) Two-dimensional TOCSY spectrum. Expansion of the region between 3.4 and 5.6 p.p.m. showing the connectivities in each monosaccharide residue. (C) Two-dimensional DQFCOSY spectrum.

Activity using different acceptors

The [alpha]-GalNAc-T activity from fetal mouse brain was tested with different artificial [beta]-D-glucuronide acceptors. As shown in Figure 4A, the aglycone structure strongly affects the acceptor activity. Both, GlcA-[beta]-MU and [alpha]-naphthyl [beta]-D-glucuronide, with bicyclic aromatic rings, were the best acceptors, while the others showed much less or virtually no activity. We also assayed the [alpha]-GalNAc-T activity using various 4-methylumbelliferyl (MU) glycosides (NeuAc-[alpha]-MU, Gal-[beta]-MU, IdoA-[alpha]-MU, Lac-[beta]-MU, Xyl-[beta]-MU, 6-SO3-GlcNAc-[beta]-MU) at 0.5 mM and 5 mM, but none had any GalNAc acceptor activity (data not shown). When GlcNAc-[beta]-MU was tested, GalNAc was transferred in [beta]-linkage to form a LacdiNAc (GalNAc-[beta]-GlcNAc[beta]-) structure, revealing a different enzymatic activity.


Figure 4. [alpha]-GalNAc-T acceptor activity of various glucuronides. Activity of [beta]-glucuronides (A) and terminal [beta]-GlcA-containing sugar molecules (B) as acceptors for the [alpha]-GalNAc-T present in FMB19 microsomal fraction. Relative activities were calculated as the percentage incorporation compared to GlcA-[beta]-MU. (A) Various glucuronides were tested for the [alpha]-GalNAc-T under standard conditions and the incorporation of [3H]GalNAc into the glucuronide acceptors was determined after C18 spin column purification as described in Materials and methods, unless otherwise stated. (B) Oligosaccharides bearing nonreducing end [beta]-GlcA residue were compared to that of GlcA-[beta]-MU. For 2AB-labeled sugars, the reaction was terminated by adding ethanol and the aliquot of soluble fraction was analyzed by HPLC as described in Materials and methods. N.D., Not detected. The results represent one of two experiments.

Since the [alpha]-GalNAc-T activity was initially identified in conjunction with the glycosaminoglycan-protein linkage region, GlcA[beta]1,3Gal[beta]1,3Gal[beta]1,4Xyl[beta]-MU (Core4-MU), chondroitin disaccharide, and hyaluronan disaccharide were also tested as potential acceptors (Figure 4B). The Core4-MU was purified from the culture medium of CHO cells incubated with Xyl-[beta]-MU as described in Materials and methods. The chondroitin or hyaluronan disaccharide was obtained by digesting chondroitin or hyaluronan with sheep testes hyaluronidase followed by labeling with 2-aminobenzamide (2AB) and then separated by charge-based HPLC fractionation. Core4-MU had 40% of the acceptor activity found for GlcA-[beta]-MU. Chondroitin disaccharide labeled with 2AB, GlcA[beta]1,3GalNAc-2AB (CS2-2AB), and the hyaluronan disaccharide unit, GlcA[beta]1,3GlcNAc-2AB (HA2-2AB), showed almost the same acceptor activities, but they were much lower (20%) than that of GlcA[beta]-MU. The N-acetylhexosamine at the reducing end of the oligosaccharide opens during the 2-AB labeling yielding an open chain. Thus, this compound may have lost its activity as compared to intact oligosaccharides in the chair conformation.

In addition to commercially available [beta]-D-glucuronides and Core4-MU, we tested a synthesized glucuronide, GlcA[beta]1,3Gal[beta]1,4GlcNAc[beta]-O-octyl ([Delta]SHNK-C8), which resembles the HNK-1 carbohydrate epitope, but lacks sulfate. This acceptor was chosen, since few other naturally occurring molecules contain terminal [beta]-GlcA residues. This compound is an efficient acceptor for in vitro sulfotransferase assays that make 3-SO3-GlcA of the HNK-1 epitope (Ong et al., 1998). Interestingly, the complex glucuronide, [Delta]SHNK-C8, showed an acceptor activity comparable to the best simple glucuronide acceptors and much higher activity than that of Core4-MU.

These results suggest that the HNK-1 epitope precursor might be one of the potential acceptors of [alpha]-GalNAc-T in naturally occurring glycoconjugates rather than chondroitin sulfate GAG-related oligosaccharides.

The effects of substrate concentrations on [alpha]-GalNAc-T are shown in Figure 5A,B. The apparent Km for UDP-GalNAc was 19 µM. The Km for artificial acceptors, GlcA-[beta]-MU and [Delta]SHNK-C8, was 0.19 mM and 0.5 mM, respectively. These values are considerably lower than those of other common glycosyltransferase acceptors which often require high mM concentrations of acceptors. This agreed with our previous results showing that [alpha]-GalNAc-capped [beta]-xyloside was the major product made when cells were incubated at low xyloside concentrations of 50 µM.


Figure 5. Effects of concentrations of acceptor sugars and UDP-GalNAc on the [alpha]-GalNAc-T present in FMB19 microsomal fraction. (A) GlcA-[beta]-MU (open squares) or [Delta]SHNK-C8 (solid squares) was added to the reaction mixture at different final concentrations. (B) UDP-GalNAc was added to the reaction mixture at different final concentrations. The insets show Lineweaver-Burk plots of the same data. Data represent one of three independent experiments.

The enzyme reaction was inhibited at higher concentration of GlcA-[beta]-MU by either the product or substrate. Therefore, all data for GlcA-[beta]-MU was collected using <1 mM of acceptor. Kinetic parameters for the [alpha]-GalNAc-T in fetal mouse brain extract are listed in Table I.

Table I. Kinetic parameters of [alpha]-GalNAc-T in fetal mouse brain extracts
Substrates Km (µM) Vmax (pmol/h/mg)
UDP-GalNAc 19 290
GlcA-[beta]-MU 190 95
[Delta]SHNK-C8 500 90

GlcA-[beta]-MU and [Delta]SHNK-C8 compete for [alpha]-GalNAc addition

We had insufficient material to confirm the GalNAc-GlcA linkage of [Delta]SHNK-C8 acceptor by 1H-NMR analysis. However, we assume that the same enzyme activity transferred an [alpha]-GalNAc to both GlcA-[beta]-MU and [Delta]SHNK-C8, since exoglycosidase digestion suggested that the GalNAc residue is transferred to GlcA in [alpha]-linkage. The products of the GalNAc-T reaction were purified by amine adsorption HPLC and digested with either [alpha]-N-acetylgalactosaminidase or [beta]-N-acetylhexosaminidase. [alpha]-N-Acetylgalactosaminidase released all of the label as free [3H]GalNAc, but [beta]-hexosaminidase did not release any label at all. [beta]-Glucuronidase digestion of the [3H]GalNAc-labeled product did not shift the elution position of the product on HPLC, suggesting that the GlcA residue is covered by the terminal GalNAc residue. To strengthen this hypothesis, we tested the GlcA-[beta]-MU for its inhibitory effects on the reaction using [Delta]SHNK-C8. At a fixed concentration of 0.5 mM [Delta]SHNK-C8, increasing GlcA-[beta]-MU concentration in the reaction mixture showed that GlcA-[beta]-MU inhibited production of [alpha]-GalNAc-[Delta]SHNK-C8 by competition of the transfer reaction, while the total amount of products remained almost the same, indicating that both acceptors were utilized by the same [alpha]-GalNAc-T (Figure 6). Therefore, it is most likely that the GalNAc was bound through [alpha]1,4-linkage to the GlcA residue of [Delta]SHNK-C8, similar to GlcA-[beta]-MU.


Figure 6. Substrate competition of the [alpha]-GalNAc-T present in FMB19 microsomal fraction. Increasing amounts of GlcA-[beta]-MU were added to each reaction mixture at a fixed concentration of [Delta]SHNK-C8 (0.5 mM). After the reaction, the products were analyzed on HPLC by injecting the same proportion of each reaction mixture. Right side of the panel: the acceptors added to the reaction mixture were shown in bar graph along with their concentration. Left side of the panel shows the chromatograms of the products made in each condition. Arrow a, product on [Delta]SHNK-C8 acceptor; arrow b, product on GlcA-[beta]-MU acceptor.

Discussion

Recent reports by us (Manzi et al., 1995; Salimath et al., 1995; Miura and Freeze, 1998) and others (Kitagawa et al., 1995b) pointed to the possibility of adding an [alpha]-GalNAc to terminal glucuronic acid residues, although physiological substrates have not yet been identified. We report here that an active enzyme capable of transferring GalNAc from UDP-GalNAc in [alpha]1-4-linkage to several glucuronides is commonly expressed in various mammalian cells and in mouse brain and that its acceptor specificity is not restricted to chondroitin oligomers as initially suggested. We also detected the same activity in adult mouse and rat brain microsomal fractions (data not shown). Characterization of the enzyme from fetal mouse brain demonstrated that Mn2+ ions are required for maximal activity and that it has a broad pH optimum with the maximum around pH 7. Acceptor specificity of the enzyme toward various glucuronides revealed that the enzyme preferred [beta]-D-glucuronides having a bicyclic aromatic ring, suggesting that the enzyme recognizes additional features of the molecule, not only the terminal GlcA residue. Interestingly, high heparan sulfate priming activity by [beta]-D-xylosides in CHO cells was shown using xylosides containing two fused aromatic rings, such as 2-naphthol-[beta]-D-xyloside and 3-estradiol-[beta]-D-xyloside (Fritz et al., 1994).

We tested potentially biologically significant acceptors for the [alpha]-GalNAc-T, including GAG-protein linkage tetrasaccharide (Core4-MU) and chondroitin/hyaluronan disaccharides as acceptors. All these molecules are related to GAG biosynthesis. Although we could not determine the apparent Km values for the acceptor because of a limited amount of Core4-MU, the relative rate for the acceptor was significantly lower than that for the artificial acceptor GlcA-[beta]-MU and nonsulfated HNK-1 precursor trisaccharide. Also, the chondroitin and the hyaluronan disaccharide labeled with 2-AB showed less acceptor activities probably due to the fact that the N-acetylhexosamine is in an open chain form.

An enzyme activity previously found in bovine serum that is capable of transferring GalNAc to chondroitin sulfate oligomers (Kitagawa et al., 1995b) is likely to be a bovine counterpart of the enzyme studied here based on the structures of products and acceptors. Since the [alpha]-GalNAc-T is localized to Golgi apparatus in M21 cells (Miura and Freeze, 1998) and is in the microsomal fractions of various cells employed in this study, the bovine enzyme found in serum would appear to be a truncated form of the membrane-bound Golgi enzyme. The previous characterization of the bovine serum [alpha]-GalNAc-T employed a series of glycoserines and chondroitin oligomers as acceptors, and the activity was referred to as UDP-GalNAc:chondro-oligosaccharide [alpha]-N-acetylgalactosaminyltransferase. Our data strongly suggest that the enzyme is capable of transferring GalNAc not only to chondro-oligosaccharides but also to the nonsulfated HNK-1 epitope-like glycoside, in addition to some simple [beta]-D-glucuronides.

Recently, a couple of interesting related observations have been reported. An [alpha]-GalNAc-T activity capable of adding a GalNAc to the linkage tetrasaccharide serine was found in mouse mastocytoma cells (Lidholt et al., 1997). Neither [beta]-linked GalNAc nor [alpha]-linked GlcNAc residues were transferred to the tetrasaccharide serine. According to Kitagawa et al., the bovine serum [alpha]-GalNAc-T showed a broad specificity toward GAG-related oligosaccharides or their serine conjugates. Also, Kitagawa et al. showed that a bovine serum [beta]-glucuronyltransferase involved in CS chain elongation does not transfer GlcA to an [alpha]-GalNAc-capped molecule, GalNAc[alpha]1-4GlcA[beta]1-3Gal[beta]1- 3Gal[beta]1-4Xyl[beta]1-O-Ser (Kitagawa et al., 1997). It has been reported that the nonreducing end structure of chondroitin sulfate chains on aggrecan is mainly terminated with [beta]-linked sulfated GalNAc (Plaas et al., 1997). This suggests that the [alpha]-GalNAc-capping is not a termination signal for chondroitin sulfate in aggrecan from Swarm Rat chondrocytes. Besides the mature chondroitin sulfate chains, the structure of immature CS chain of [alpha]-thrombomodulin ([alpha]-TM) known as a 'part-time" proteoglycan has been reported (Nadanaka et al., 1998). They found that the [alpha]-TM carries the linkage tetrasaccharide at the GAG attachment site. Here again, the [alpha]-GalNAc termination of [beta]-GlcA was not detected, indicating that the [alpha]-GalNAc-capping chain is not the termination signal for CS biosynthesis in this 'part-time" proteoglycan. Combining these results with ours showing that the [alpha]-GalNAc-T in FMB19 prefers [Delta]SHNK-C8 oligosaccharide to the GAG-related oligosaccharides, the [alpha]-GalNAc-T may not be involved in conventional GAG biosynthesis.

When [beta]-xylosides are incubated with melanoma or CHO cells at low concentrations (5-100 µM), the predominant product is an [alpha]-GalNAc-capped xyloside (Salimath et al., 1995). This preference at low concentration of [beta]-xylosides agrees with our results showing very low apparent Km values of acceptors. The modification of GlcA by terminal [alpha]-GalNAc does not seem to be an artifact, but there have been no reports on naturally occurring glycoconjugates carrying this structure. Since our results suggested that other GlcA-terminated molecules besides GAGs could be [alpha]-GalNAc-capped, especially glucuronylated lactosamine, we tried to identify a terminal [alpha]-GalNAc structure in glycoproteins, glycolipids, or proteoglycans. Using Lec2 cells, a mutant of CHO cell, transfected with [beta]-glucuronosyltransferases, GlcAT-I (Wei et al., 1999) and GlcAT-P (Terayama et al., 1997), and parent Lec2 cells, PNGaseF-released [3H]mannose-labeled oligosaccharides from total cell lysates were tested for the presence of the terminal [alpha]-GalNAc structure. Since the Lec2 cells have a markedly reduced transport of CMP-sialic acid into the Golgi, they expresses glycoproteins without terminal sialic acids. Using these cell lines increases the proportion of oligosaccharides that are acceptors for GlcA residues. The released sugar chains were analyzed on HPLC before or after treatments with [alpha]-N-acetylgalactosaminidase and/or [beta]-glucuronidase. We found that anionic sugar chains were increased significantly in the transfected cells compared to those of parent cells, but no evidence of [alpha]-GalNAc-capping was detected (unpublished observations). In addition, glycolipid extracts from an adult rat brain were also analyzed on HPLC for the [alpha]-GalNAc-capped species after releasing the sugars with ceramide glycanase followed by 2-AB labeling. Using exoglycosidase digestion as mentioned above, we could not find any [alpha]-GalNAc-terminated molecules (unpublished observations).

There are no reports of this structure on natural glycoconjugates so far. However, some studies suggest the presence of [alpha]-GalNAc-terminated molecules other than known structures (Brooks and Leathem, 1995; Rye et al., 1998). Using Helix pomatia (HPA) lectin, Brooks and Leathem concluded that the breast cancer-derived glycoproteins containing a terminal [alpha]-GalNAc residue cannot simply be blood group A glycans or Tn epitopes ([alpha]-GalNAc-O-Ser/Thr) (Brooks and Leathem, 1995). We found that [alpha]-GalNAc-capped penta- or heptasaccharyl xylosides are retarded on a HPA lectin affinity column (Miura and Freeze, 1998). Thus, it might be possible to purify [alpha]-GalNAc-terminated glycoconjugates on a HPA column. We also tried this approach in both melanoma and CHO cells, but could not detect such molecules. Further studies are needed to identify the role of this [alpha]-GalNAc-T in natural glycoconjugates biosynthesis. If terminal [alpha]-GalNAc is found in naturally occurring glycoconjugates, it would probably be in an undescribed pathway. Alternatively, [alpha]-GalNAc may only be a transient intermediate in such a pathway.

Materials and methods

Materials

UDP-[3H]GalNAc was purchased from American Radiolabeled Chemicals, St. Louis, MO. Jack bean [beta]-N-acetylhexosaminidase and chicken liver [alpha]-N-acetylgalactosaminidase were purchased from Oxford GlycoSciences. Bovine liver [beta]-glucuronidase, Silica gel (15-40 µ), and Xyl-[beta]-MU were purchased from Sigma. GlcA-[beta]-MU and UDP-GalNAc were from Calbiochem, San Diego, CA. [beta]-D-Glucuronides were obtained from Sigma unless otherwise stated. C18 silica gels (Bonded C18 reversed phase silica gel and UNIBOUND C18 SPICE Tube) were from Analtech, Inc., Newark, DE. [Delta]SHNK-C8 was prepared as previously described (Ding et al., 1998). Chicken liver [alpha]-N-acetylgalactosaminidase and jack bean [beta]-N-acetylhexosaminidase were purchased from Oxford GlycoSciences, UK. Bovine liver [beta]-glucuronidase was obtained from Sigma.

Cell lines

The human hepatoma cells C3A and normal human fibroblast were from American Type Culture Collection (ATCC). Chinese hamster ovary (CHO) cells were kindly provided by Dr. J.D.Esko, University of California. Lec2 cells transfected with glycoprotein-specific glucuronosyl transferase (Ong et al., 1998) and glucuronosyl transferase I were kindly provided by Dr. M.Fukuda, The Burnham Institute, CA, and Dr. J.D.Esko, University of California, CA, respectively. Human kidney epithelial cells 293T were provided by Dr. J.C.Reed, The Burnham Institute, CA. Monkey kidney COS-1 cell was provided by Dr. M.Fukuda, The Burnham Institute, CA. Human melanoma cells M21 were provided by Dr. J.M.Trent, University of Michigan, Ann. Arbor, MI. Normal human fibroblasts were obtained from Advanced Tissue Sciences, Inc., La Jolla, CA.

Preparation of microsomal fractions

Female pregnant CD1 strain mouse was sacrificed at embryonic day 19. Brains from the fetal mice were taken and used as a source for the fetal mouse brain microsomes. Brains (0.88 g wet) taken from embryo were homogenized in 2.4 ml of 10 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose using a Dounce homogenizer. The homogenates were centrifuged at 10,000 × g for 10 min. The supernatant was centrifuged at 105,000 × g for 60 min. The supernatant was discarded, and the pellets were suspended in 600 µl of 10 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose and stored at -80°C before use at protein concentration of 15 mg/ml. Microsomal fractions of various cultured cell lines were obtained in a same manner.

Purification of Core4-MU xylosides from culture medium

The CHO mutant cell line, Lec2, was incubated with 1 mM Xyl-[beta]-MU for 3 days in [alpha]-MEM supplemented with 10% fetal calf serum. Fifty milliliters of the culture medium was passed through a QAE column, and the fraction that did not bind to the column and the PBS washings were collected. The material was applied to a column of UNIBOUND C18 SPICE Tube (Analtech). The column was washed with water, and the bound material was eluted with 5 ml of 50% MeOH. The eluate was dried in a SpeedVac concentrator (Savant). The residue was dissolved in 20 µl of 30% AcOH-DMSO and diluted with 0.4 ml of acetonitrile. The solution was applied to a silica gel spin column, packed in a 0.2 µm pore sized PVDF centrifuge filter (Whatman Inc.) and then equilibrated with acetonitrile. The column was washed with acetonitrile (400 µl × 3), and 4% water-acetonitrile (400 µl × 3). Carbohydrate-related compounds were eluted with 50% aqueous acetonitrile, and the eluate was dried. The sample was dissolved in 50% aqueous acetonitrile and subjected to amine adsorption HPLC using a column of Microsorb-MV NH2 (4.6 × 250 mm) as described below. The eluate was monitored with a fluorescence detector (DYNAMAX FL-1, Rainin) and 0.5 ml fractions were collected. The fractions containing fluorescence were subjected to mass analysis using a KOMPACT MALDI I (linear time-of-flight, Kratos Analytical, United Kingdom) mass spectrometer. A fraction containing a compound with m/z of 809.0 (calculated m/z 808.23 for Core4-MU) was identified as GlcA[beta]Gal[beta]Gal[beta]Xyl[beta]-MU, Core4-MU, in conjunction with exo-glycosidase digestion (data not shown). The purified compound was tested for [beta]-glucuronidase susceptibility and the product was analyzed by HPLC to show comigration with authentic [3H]-labeled Core4-MU. [beta]-Glucuronidase digestion was carried out in sodium acetate buffer, pH 5, using the enzyme at 20-50 unit/µl.

N-Acetylgalactosaminyltransferase assay

The standard reaction was performed in a final volume of 10 µl containing 50 mM MES, pH 6.7, 0.5 mM GlcA-[beta]-MU, 10 µM of 0.125 µCi UDP-[3H]GalNAc, 10 mM MgCl2, 10 mM MnCl2, 0.1% Triton X-100, and 10-20 µg of microsomal protein for 1 h at 37°C. The reaction was terminated by adding 290 µl of 20 mM NaHCO3 containing 10 mM EDTA followed by heating at 100°C for 3 min. The reaction mixtures were centrifuged for 10 min in an Eppendorf centrifuge at maximal speed. Since most of the acceptors have a hydrophobic aglycone, recovery of products was carried out using reversed-phase C18 extraction. The supernatant was applied on a C18 spin column preactivated with MeOH and equilibrated with 20 mM NaHCO3 containing 10 mM EDTA. The column was washed three times with 300 µl of 20 mM NaHCO3 containing 10 mM EDTA, and the bound material was eluted with 300 µl of 50% MeOH (twice). The combined eluates were counted for radioactivity. To determine relative rates of some acceptors, the [alpha]-GalNAc-T reaction was terminated by adding 390 µl of ice-cold ethanol and the solution was centrifuged at 4°C to remove the precipitates;. 100 µl of the supernatant was dried and subjected to glycosidase digestion followed by HPLC analysis.

Preparation and labeling of chondroitin and hyaluronan oligomers

Twenty milligrams of chondroitin (Seikagaku Corp., Japan) or hyaluronic acid (Sigma) was hydrolyzed with 2000 U of sheep testes hyaluronidase (Sigma) in 1 ml reaction mixture containing 50 mM sodium acetate, pH 5.2, and 50 mM NaCl. After termination of the reaction by heating, the mixture was concentrated to 100 µl and applied to a Sephadex G-25 column (0.7 × 48 cm). Included materials were combined and lyophilized. This material was labeled with 2-aminobenzamide (2AB) as described previously (Bigge et al., 1995). After the labeling reaction, the oligosaccharide-2AB conjugates were purified using a silica gel spin column, a centrifuge filter (PVDF filter media, Whatman Inc.) packed with 200 µl bed volume of silica gel (15-40 µ, Sigma). The reaction mixture (10 µl) was diluted with 400 µl of acetonitrile, applied on a silica gel spin column which was pre-washed with water and then equilibrated with acetonitrile. The column was washed with 4 × 400 µl of acetonitrile, and 4 × 400 µl of 4% water in acetonitrile. The labeled samples eluted with water were dried. The oligosaccharides labeled with 2AB were fractionated by HPLC based on their charges as described below.

High performance liquid chromatography

Anionic and neutral oligosaccharides were eluted at 0.5 ml/min from a Microsorb-MV column (NH2), 4.5 mm × 25 cm, using an aqueous ammonium formate (pH 6) and acetonitrile gradient: 0-5 min, 10 mM ammonium formate and 80% acetonitrile; 5-45 min, 10 mM ammonium formate and acetonitrile decreased from 80% to 40%; 45-75 min, ammonium formate increased from 10 mM to 150 mM and acetonitrile maintained at 40%; 75-90 min, ammonium formate increased to 250 mM and acetonitrile decreased to 0%. The radioactivity was counted with an on-line [beta]-counter.

Mass spectrometry

MALDI-MS experiments were performed with KOMPACT MALDI I (linear time-of-flight, Kratos Analytical, UK) mass spectrometer in the positive ion mode (nitrogen laser light; [lambda] = 337 nm, pulse; 3 ns). Samples eluted from the HPLC was collected, and dried using a SpeedVac. The drying was repeated after adding water to remove volatile salts. 0.5 µl (10-100 pmol) of the xylosides dissolved in 50% acetonitrile were applied to the stainless steel target covered with crystals of a mixture of 2,5-dihydroxy benzoic acid and 1-hydroxy isoquinoline (Mohr et al., 1995) and dried.

Enzyme digestions

Aliquots containing 5000-10,000 c.p.m. of each sample were dried after the [alpha]-GalNAc-T reaction, were digested with 0.1 U for [beta]-N-acetylhexosaminidase and 5 mU for [alpha]-N-acetylgalactosaminidase for 9 h at 37°C in 20 µl buffer supplied from the manufacturer. Samples for HPLC analysis were diluted with an equal volume of acetonitrile and heated at 100°C for 2 min, followed by centrifugation for 10 min to remove any precipitate.

1H-NMR spectroscopy

In order to isolate enough quantity of the product obtained by treatment of GlcA-[beta]-MU with [alpha]-GalNAc-T, a large-scale transferase reaction was carried out (1.6 ml total volume, containing 1 mM acceptor, 1.5 mM donor, UDP-GalNAc, and 6 mg FMB19 microsomal protein) for 14 h at 37°C. At the end of the reaction, the mixture was heated at 100°C for 10 min and centrifuged to remove any precipitate. The supernatant was concentrated and subjected to a C18 and silica gel spin column as described above. The dried material was dissolved in 50 µl of 50% aqueous acetonitrile and fractionated by an HPLC system. Nearly 20 µg of disaccharide was obtained and used for NMR analysis. The sample was repeatedly exchanged in D2O (99.96%, Aldrich Chemical Co.) with intermediate lyophilization, and finally dissolved in 40 µl of 99.996% D2O. The standard was dissolved in DMSO-d6. Spectra were recorded on a Varian Innova 500 MHz NMR spectrometer at the UCSD Glycobiology Core Facility. Proton 1D NMR spectra were obtained using presaturation of the HOD peak. TOCSY 2D spectra were obtained using a 10 kHz MLEV17 spin lock of either 50 or 100 msec duration. Double-quantum-filtered COSY (DQFCOSY) spectra were obtained using 200 complex t1-data points having 16 scans each. ROESY 2D data were obtained using a 2 kHz pulsed spinlock of 200 msec duration, using 96 scans per 160 complex t1-data points. All 2D spectra were obtained in a phase-sensitive mode using hypercomplex sampling in t1, and included presaturation of the HOD resonance. Line broadening (0.2 Hz) was applied to 1D spectra, while 2D spectra were weighted with Gaussian functions. Chemical shifts were actually measured relative to the residual HOD peak at 4.80 p.p.m.. Assignments were made by comparing with data in the literature and with the standard GlcA-[beta]-MU analyzed in parallel.

Acknowledgments

Supported by American Cancer Society Grant BE242 and California Tobacco Disease Related Research Program 4IT-0225 (H.H.F.), and a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists (Y.M.)

Abbreviations

MU, 4-methylumbelliferyl; GalNAc, N-acetyl-D-galactosamine; Man, mannose; NeuAc, N-acetylneuraminic acid; Gal, galactose; IdoA, iduronic acid; Lac, lactose; Xyl, xylose; GlcNAc, N-acetyl-D-glucosamine; FMB19, fetal mouse brain at day 19, [Delta]SHNK-C8, GlcA[beta]-1,3-Gal[beta]-1,4-GlcNAc[beta]-O-octyl; HPLC, high-performance liquid chromatography; DMSO, dimethylsulfoxide; TOCSY, totally scalar correlated spectroscopy; DQFCOSY, double-quantum-filtered correlation spectroscopy; ROESY, rotating frame Overhauser enhancement spectroscopy; CS, chondroitin sulfate; HA, hyaluronic acid; Core4-MU, GlcA[beta]-1,3-Gal[beta]-1,3-Gal[beta]1, 4-Xyl[beta]-MU; HPA, Helix pomatia agglutinin; PNGaseF, peptide-N-glycosidase F

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