©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

(Received for publication, May 23, 1995; and in revised form, July 13, 1995)

Hiroshi Kitagawa Yukako Tanaka Kazunori Tsuchida Fumitaka Goto (1) Tomoya Ogawa (1) (2) Kerstin Lidholt (3) Ulf Lindahl (3) Kazuyuki Sugahara (§)

From the  (1)Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, Japan, RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama, 351-01, Japan, the (2)Graduate School for Agriculture and Life Science, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan, and the (3)Department of Medical and Physiological Chemistry, University of Uppsala, The Biomedical Center, S-751 23 Uppsala, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

During the course of a study to elucidate the role of modification of the common polysaccharide-protein linkage structure, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, in biosynthetic sorting mechanisms of the different sulfated glycosaminoglycan chains, a novel N-acetylgalactosamine (GalNAc) transferase was discovered in fetal bovine serum. The enzyme catalyzed the transfer of [^3H]GalNAc from UDP-[^3H]GalNAc to linkage tetrasaccharide and hexasaccharide serines synthesized chemically and to various regular oligosaccharides containing terminal D-glucuronic acid (GlcA), which were prepared from chondroitin and chondroitin sulfate using testicular hyaluronidase digestion. The labeled products obtained with the linkage tetra- and hexasaccharide serines and with the tetrasaccharide (GlcAbeta1-3GalNAc)(2) were resistant to digestion with chondroitinase AC-II and beta-N-acetylhexosaminidase but sensitive to alpha-N-acetylgalactosaminidase digestion, indicating that the enzyme is an alpha-N-acetylgalactosaminyltransferase. This finding is in contrast to that of Rohrmann et al. (Rohrmann, K., Niemann, R., and Buddecke, E.(1985) Eur. J. Biochem., 148, 463-469), who reported that a corresponding product was susceptible to digestion with beta-N-acetylhexosaminidase. The presence of a sulfate group at C4 of the penultimate GalNAc or Gal units markedly inhibited the transfer of GalNAc to the terminal GlcA, while a sulfate group at C6 of the GalNAc had little effect on the transfer. Moreover, a slight but significant transfer of GalNAc was observed to an oligosaccharide containing terminal 2-O-sulfated GlcA as acceptor, whereas no incorporation was detected into oligosaccharides containing terminal unsaturated or 3-O-sulfated GlcA units. These results suggest that this novel serum enzyme is a UDP-GalNAc:chondro-oligosaccharide alpha1-3- or 1-4-N-acetylgalactosaminyltransferase. The possibility of involvement of this enzyme in glycosaminoglycan biosynthesis is discussed.


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, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (for reviews, see (1) and (2) ). Heparin/heparan sulfate is synthesized once GlcNAc is transferred to the common linkage region, while chondroitin sulfate is formed if GalNAc is first added. The two distinct transferases, which catalyze the transfer of GlcNAc or GalNAc, (^1)respectively, to the common linkage region are the key enzymes that determine the type of glycosaminoglycans to be synthesized. The transferases for the first GlcNAc and GalNAc residues are thought to be different from the glycosyltransferases catalyzing the elongation steps, which transfer GlcNAc or GalNAc to the corresponding repeating disaccharide region. Molecular mechanisms are unknown, however, for the synthesis of different glycosaminoglycans on the common linkage region.

We have been investigating the structure of the linkage region of various glycosaminoglycans to search for possible structural differences that may determine the characteristics of the glycosaminoglycan species to be synthesized. These structural studies revealed that sulfation of C6 on both Gal residues and C4 of Gal adjacent to GlcA was characteristic of chondroitin sulfate(3, 4, 5, 6, 7) . Sulfation of C4 of the Gal residue was also demonstrated in the linkage region of bovine aorta dermatan sulfate(8) . Sulfated Gal residues have not been found to date in the linkage region of heparin or heparan sulfate(9, 10) . In view of these structural variations in the linkage region, we investigated the effects of sulfation in the linkage region on the specificity of GalNAc transferase, which is involved in the biosynthesis of chondroitin sulfate using chemically synthesized linkage tetrasaccharide serines, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-sulfate)beta1-3Gal beta1-4Xylbeta1-O-Ser, and hexasaccharide serines, GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, GlcAbeta1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, and GlcAbeta1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser, as acceptor substrates(11, 12, 13) . During studies to characterize the enzyme products, we unexpectedly found that the products contained the transferred [^3H]GalNAc alpha-linked to the nonreducing terminal GlcA of the acceptor substrates employed. Since the occurrence of such an enzyme has not been previously documented, we investigated its substrate specificity further using a series of oligosaccharides derived from various chondroitin sulfate isomers.


EXPERIMENTAL PROCEDURES

Materials

Fetal bovine serum was obtained from ICN Biomedicals Japan (Osaka). UDP-[^3H]GalNAc (6.3 Ci/mmol) and unlabeled UDP-GalNAc were purchased from DuPont NEN and Sigma, respectively. The following linkage tetrasaccharide and hexasaccharide serines were synthesized chemically(11, 12, 13) : GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser, GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, GlcAbeta1-3GalNAc(4sulfate)beta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, and GlcAbeta1- 3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylbeta1-OSer. Unsaturated linkage tetrasaccharide and hexasaccharide serines, DeltaGlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and DeltaGlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, were isolated from porcine intestinal heparin and rat chondrosarcoma chondroitin 4-sulfate as previously described(3, 9) . Globo-N-tetraose (GalNAcbeta1-3Galalpha1-4Galbeta1-4Glc) and lacto-N-fucopentaose I (Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) were purchased from BioCarb Chemicals (Lund, Sweden). Apomucin was prepared from the bovine submaxillary gland mucin as described by Sugiura et al.(14) . Polymeric chondroitin, sheep testis hyaluronidase, Jack bean beta-N-acetylhexosaminidase, Arthrobacter aurescens chondroitinase AC-II, and Acremonium sp. alpha-N-acetylgalactosaminidase were obtained from Seikagaku Corp. (Tokyo). Even-numbered regular chondro-oligosaccharides (GlcAbeta1-3GalNAc)(2), (GlcAbeta1-3GalNAc)(3), and (GlcAbeta1-3GalNAc)(4) were prepared by hyaluronidase digestion of chondroitin followed by gel filtration on a Bio-Gel P-10 column (1.6 times 95 cm), and then HPLC on an amine-bound silica column as previously described(15) . Other sulfated tetra- and hexasaccharides used as acceptors were isolated by HPLC after hyaluronidase digestion of whale cartilage chondroitin sulfate A, shark cartilage chondroitin sulfate D, king crab cartilage chondroitin sulfate K, and bovine bronchial cartilage chondroitin sulfate and were structurally characterized enzymatically and also by 500-MHz ^1H NMR spectroscopy when required. (^2)

GalNAc Transferase Assay

Incubation mixtures contained the following constituents in a total volume of 35 µl: 1 nmol of a linkage tetra- or a hexasaccharide serine, a chondro-oligosaccharide, or 300 µg of chondroitin, 8.57 µM UDP-[^3H]GalNAc (5.28 times 10^5 dpm), 50 mM MES buffer, pH 6.5, 10 mM MnCl(2), 10 mM MgCl(2), 171 µM ATP, and 18 µl of fetal bovine serum. Reaction mixtures were incubated at 37 °C for 21 h, and the reactions were terminated by addition of 35 µl of cold 10% (w/v) trichloroacetic acid. The mixtures were centrifuged for 5 min in an Eppendorf centrifuge at top speed. The supernatant (65 µl) was extracted twice with 1 ml of ether, and the lower aqueous phase was subjected to chromatography on a column (1 times 106 cm) of Sephadex G-25 (superfine) equilibrated and eluted with 0.25 M NH(4)HCO(3)-7% 1-propanol. Fractions (1 ml each) were collected at a rate of 4.8 ml/h and analyzed for radioactivity.

Characterization of the Reaction Products

Isolation of the products from GalNAc transferase reactions was carried out by gel filtration on a Sephadex G-25 column (1 times 106 cm), which was eluted with 0.25 M NH(4)HCO(3), 7% 1-propanol. Digestion of the isolated products with chondroitinase AC-II, beta-N-acetylhexosaminidase, or alpha-N-acetylgalactosaminidase was performed essentially according to the instructions provided by the manufacturer. Briefly, labeled products (about 1.5 pmol) corresponding to 2000-2500 dpm were incubated at 37 °C overnight with 100 mIU of chondroitinase AC-II in a total volume of 40 µl of 30 mM sodium acetate, pH 5.5, or with 500 mIU of beta-N-acetylhexosaminidase or 52 mIU of alpha-N-acetylgalactosaminidase in a total volume of 25 µl of 50 mM sodium citrate, pH 4.5, respectively. The enzyme digest was diluted to 200 µl with 0.25 M NH(4)HCO(3), 7% 1-propanol and analyzed using the same Sephadex G-25 column as above.

Substrate Competition Experiments

The competition experiments between the chondro-oligosaccharide (GlcAbeta1-3GalNAc)(2) and either lacto-N-fucopentaose I or apomucin for the alpha-N-acetylgalactosaminyltransferase in the heat-treated serum (50 °C for 60 min) were performed in the reaction mixture described above. Incubation mixtures contained either 1 nmol of the lacto-N-fucopentaose I and 10 nmol of the chondro-oligosaccharide (GlcAbeta1-3GalNAc)(2) or 1 nmol of the chondro-oligosaccharide (GlcAbeta1-3GalNAc)(2) and 300 µg of apomucin. After the reaction, the radioactive products were analyzed using a Sephadex G-25 column as described above. The products obtained with two acceptors in each competition experiment were well separated by gel filtration.


RESULTS

Serum GalNAc Transferase Activity toward the Common Linkage Region

Fetal bovine serum was tested as an enzyme source for GalNAc transferase since several biosynthetic enzymes for chondroitin sulfate and heparin/heparan sulfate have been demonstrated to occur at high concentrations in serum(16, 17, 18, 19) , and the serum enzymes are stable and occur in soluble forms in nature. Fetal bovine serum also contains an enzyme that catalyzes the transfer of [^3H]GalNAc from UDP-[^3H]GalNAc to polymeric chondroitin(20) . In this study, we first investigated the role of C4 sulfation of the Gal residue linked to GlcA in the linkage region in the chondroitin sulfate biosynthesis using a nonsulfated tetrasaccharide linkage region GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and a sulfated counterpart GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser as acceptors in the serum GalNAc transferase assay. The former was a good acceptor for GalNAc transferase, and the reaction product was eluted at a position corresponding to a pentasaccharide upon Sephadex G-25 column gel filtration (Fig. 1, solid lines). In contrast, the latter compound gave an elution profile similar of the control (Fig. 1, broken lines) and therefore could not serve as an acceptor substrate when assayed under the same conditions as used for the nonsulfated compound. The results indicate that C4 sulfation of the Gal completely precludes the transfer of the first GalNAc residue to the linkage tetrasaccharide core.


Figure 1: Gel filtration analysis of GalNAc transferase reaction products obtained with the tetrasaccharide serines as acceptors. The GalNAc transferase reaction was carried out in the presence (solid line) or absence (broken lines) of the linkage tetrasaccharide serine GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser as an acceptor as described under ``Experimental Procedures.'' The sulfated tetrasaccharide serine GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser was also tested as an acceptor, the pattern of which was indistinguishable from that of the control (broken lines). Effluent fractions were analyzed for radioactivity.



To assess the effects of C4 sulfation of the Gal and/or the first GalNAc residue on the transferase reaction of the second GalNAc residue, a nonsulfated, a monosulfated, and a disulfated hexasaccharide serine were used as acceptor substrates for the serum enzyme. As shown in Fig. 2, the nonsulfated compound GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser was the best substrate (A) followed by the disulfated compound GlcAbeta1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1- 3Galbeta1-4Xylbeta1-O-Ser (C), while little incorporation into the monosulfated compound GlcAbeta1-3GalNAc(4-sulfate)beta1- 4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (B) was observed. These results indicate that C4 sulfation of the first GalNAc residue is inhibitory to the transfer of the second GalNAc residue and that C4 sulfation of the Gal residue also has some regulatory effects on the reaction. All the above results seemed to suggest that fetal bovine serum contains GalNAc transferase(s) involved in the chain initiation and elongation of chondroitin.


Figure 2: Gel filtration analysis of GalNAc transferase reaction products obtained with the hexasaccharide serines as acceptors. The linkage hexasaccharide serines GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (A), GlcAbeta1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (B), or GlcAbeta1- 3GalNAc(4-sulfate)beta1-4GlcAbeta1-3Gal(4-sulfate)beta1-3Galbeta1-4Xylbeta1O-Ser (C) were tested as acceptors for GalNAc transferase in the serum as described under ``Experimental Procedures.'' Effluent fractions were analyzed for radioactivity.



Differential Thermostability of GalNAc Transferases in Serum

Previously, Rohrmann et al.(21) proposed that at least two GalNAc transferase activities exist in the microsomal fraction of calf arterial tissue. One, designated GalNAc transferase I (GalNAc T-I), catalyzed transfer of the first GalNAc residue immediately adjacent to the linkage region. The other, designated GalNAc transferase II (GalNAc T-II), was involved in polymerization of the chondroitin sulfate chain. In addition, they also showed that these two enzymes differed in their temperature stability; the former was stable while the latter was labile on heat treatment at 50 °C for 60 min. Therefore, we attempted to clarify whether the same holds true for the serum enzymes. Fetal bovine serum was treated at 50 °C for 60 min, and GalNAc T-I and -II were assayed using the nonsulfated tetrasaccharide serine GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and polymeric chondroitin as acceptors, respectively. In addition, the nonsulfated hexasaccharide serine GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser was also used as an acceptor. GalNAc T-I measured with the tetrasaccharide serine retained its full activity and even exhibited elevated activity (2.6-fold), while the residual activity of the GalNAc T-II measured with polymeric chondroitin was less than 5% of that obtained with the non-heat-treated serum. Surprisingly, an enzyme that utilized the nonsulfated hexasaccharide serine as an acceptor was stable to the heat treatment and even showed elevated activity (3.3-fold). Although the reason why the heat treatment of the serum resulted in the apparent elevation of the GalNAc T-I activity is unclear, it was most likely due to the inactivation of serum nucleotide pyrophosphatase and phosphatase, which hydrolyze the donor substrate UDP-GalNAc to GalNAc 1-phosphate and GalNAc, respectively. These results suggest that the GalNAc transferase, which utilized the tetrasaccharide and the hexasaccharide serines, is different from that which utilized polymeric chondroitin and that fetal bovine serum contains at least two GalNAc transferase activities as in the case of the microsomal enzymes from calf arterial tissue.

Demonstration of the alpha-Anomeric Configuration of the Incorporated GalNAc into Tetrasaccharide and Hexasaccharide Serines

To investigate the structure of the reaction products obtained by incubation of the non-heat-treated serum enzyme with the tetrasaccharide and the hexasaccharide serines, aliquots of the reaction products were treated with chondroitinase AC-II, beta-N-acetylhexosaminidase, or alpha-N-acetylgalactosaminidase, and the samples were analyzed by gel filtration on a Sephadex G-25 column. The products were compared with those obtained with polymeric chondroitin as an acceptor (Fig. 3). Unexpectedly, none of the [^3H]GalNAc transferred to the nonsulfated tetrasaccharide serine was released as free [^3H]GalNAc either by chondroitinase AC-II (panel A-a in Fig. 3) or by beta-N-acetylhexosaminidase (panel A-b). In contrast, 100 or 90% of the [^3H]GalNAc transferred to polymeric chondroitin was released by either one of these enzymes (panels C-a and C-b). Surprisingly, the reaction product obtained with the tetrasaccharide serine as an acceptor was sensitive to digestion with alpha-N-acetylgalactosaminidase yielding quantitatively a ^3H-labeled peak at the position of free [^3H]GalNAc (panel A-c), whereas that obtained with polymeric chondroitin was insensitive (panel C-c). The radiolabeled product obtained with the nonsulfated hexasaccharide serine was not degraded by beta-N-acetylhexosaminidase (panel B-b), whereas the ^3H radioactivity was almost completely released by alpha-N-acetylgalactosaminidase as free [^3H]GalNAc (panel B-c). When the transferase reaction product was digested with chondroitinase AC-II, a ^3H-labeled oligosaccharide, most likely a trisaccharide GalNAcalpha1-GlcAbeta1-3GalNAc, was generated (panel B-a). It should be noted that authentic GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, which was prepared by beta-glucuronidase treatment of the chemically synthesized hexasaccharide serine, could be digested with either chondroitinase AC-II or beta-N-acetylhexosaminidase, as judged by HPLC (data not shown). These results indicate that the enzyme in non-heat-treated serum, which utilized the tetrasaccharide and the hexasaccharide serines as acceptor substrates, was exclusively an alpha-N-acetylgalactosaminyltransferase.


Figure 3: Enzymatic characterization of the anomeric configuration of the transferred GalNAc using chondroitinase AC-II, beta-N-acetylhexosaminidase, or alpha-N-acetylgalactosaminidase. The GalNAc transferase reaction was conducted using non-heat-treated serum as an enzyme source, and the linkage tetrasaccharide serine GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (A), the linkage hexasaccharide serine GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (B), or polymeric chondroitin (C) was used as an acceptor. Each product was then isolated by gel filtration and subjected to treatment with chondroitinase AC-II (a), beta-N-acetylhexosaminidase (b), or alpha-N-acetylgalactosaminidase (c) as described under ``Experimental Procedures.''



alpha- and beta-N-Acetylgalactosaminyltransferase Activities toward Even-numbered Nonsulfated Regular Chondro-oligosaccharides

In view of the specificity of the GalNAc transferase toward the linkage tetra- and hexasaccharide serines, the acceptor specificity of the enzyme in the non-heat-treated serum was determined toward even-numbered regular chondro-oligosaccharides (GlcAbeta1-3GalNAc)(2), (GlcAbeta1-3GalNAc)(3), and (GlcAbeta1-3GalNAc)(4). The serum enzymes utilized all three oligosaccharides for GalNAc transferase reactions, although no obvious size-dependent incorporation was observed (Fig. 4). Since both alpha-N-acetylgalactosaminyltransferase and beta-N-acetylgalactosaminyltransferase are present in bovine serum as described above, the reaction products were examined in terms of their sensitivity to alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase, respectively. As shown in Fig. 4, the product obtained with (GlcAbeta1-3GalNAc)(2) was completely digested with alpha-N-acetylgalactosaminidase but not with beta-N-acetylhexosaminidase, suggesting that GalNAc had been transferred to (GlcAbeta1-3GalNAc)(2) exclusively through an alpha-linkage. In contrast, 42 and 58% of the product obtained with (GlcAbeta1-3GalNAc)(3) was sensitive to digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase, respectively. Similarly, 67 and 33% of the product obtained with (GlcAbeta1-3GalNAc)(4) was sensitive to these enzymes, respectively. These results indicate that the reaction products with (GlcAbeta1-3GalNAc)(3) and (GlcAbeta1-3GalNAc)(4) were formed by the action of both alpha-N- and beta-N-acetylgalactosaminyltransferases when non-heat-treated serum was used as an enzyme source. It should be emphasized that the regular tetrasaccharide could not serve as an acceptor for beta-N-acetylgalactosaminyltransferase despite its presence in the serum.


Figure 4: Analysis of the anomeric configuration of the GalNAc transferred onto the regular chondro-oligosaccharides. Chondro-oligosaccharide (GlcAbeta1-3GalNAc)(2) (A), (GlcAbeta1-3GalNAc)(3) (B), or (GlcAbeta1-3GalNAc)(4) (C) was used as an acceptor substrate for the GalNAc transferase reaction with the non-heat-treated serum. Each product was then purified and characterized in terms of the anomeric configuration of the transferred [^3H]GalNAc using either alpha-N-acetylgalactosaminidase or beta-N-acetylhexosaminidase as described under ``Experimental Procedures.''



As mentioned above, GalNAc incorporation into acceptor substrates through an alpha- or a beta-linkage could readily be distinguished by the differential thermostability of these two enzymes. Accordingly, the products obtained by incubation of the heat-treated serum with (GlcAbeta1-3GalNAc)(2), (GlcAbeta1-3GalNAc)(3) and (GlcAbeta1-3GalNAc)(4) were tested in terms of their sensitivity to digestion with alpha-N-acetylgalactosaminidase. The [^3H]GalNAc-labeled products obtained with all three acceptors were completely digested with alpha-N-acetylgalactosaminidase (data not shown). Thus, the heat treatment was useful to preclude the effect of the beta-N-acetylgalactosaminyltransferase activity in bovine serum.

Discrimination of the alpha-N-Acetylgalactosaminyltransferase from the Known Enzymes by Substrate Competition Studies

To investigate the novelty of the alpha-N-acetylgalactosaminyltransferase, substrate competition experiments were carried out. To our knowledge, three other alpha-N-acetylgalactosaminyltransferases have so far been investigated in detail. One of these enzymes, which is involved in the synthesis of Forssman antigen GalNAcalpha1-3GalNAcbeta1-3Galalpha1-4Galbeta1-4Glc(22) , was clearly different from the alpha-GalNAc T described in this study based on its marginal activity in heat-treated serum as detected with globo-N-tetraose (GalNAcbeta1-3Galalpha1-4Galbeta1-4Glc) (Table 1). The activity of another alpha-N-acetylgalactosaminyltransferase, blood group A-specific alpha-GalNAc transferase(23) , was detected even in heat-treated serum using lacto-N-fucopentaose I (Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) as an acceptor. However, the GalNAc transfer to lacto-N-fucopentaose I was not inhibited by (GlcAbeta1-3GalNAc)(2) as examined by gel filtration of the transferase reaction products on Sephadex G-25 (Table 1), indicating that the alpha-GalNAc T in question is different from blood group A-specific alpha-GalNAc transferase. It was also distinguished from the polypeptide alpha-GalNAc transferase responsible for the biosynthetic initiation of O-linked oligosaccharides, which catalyzes the transfer of an alpha-GalNAc to a serine or a threonine residue on the protein acceptor(14) , since the GalNAc transfer to (GlcAbeta1-3GalNAc)(2) was not inhibited by apomucin prepared from bovine submaxillary mucin (Table 1). Thus, the alpha-GalNAc T demonstrated in this study appears to be a hitherto unreported new transferase.



Characterization of Positional Specificity of the alpha-N-Acetylgalactosaminyltransferase Using the Various Oligosaccharides

To further characterize the substrate specificity of the alpha-N-acetylgalactosaminyltransferase in heat-treated serum, a variety of acceptor substrates prepared from chondroitin and chondroitin sulfates were employed, and the results are summarized in Table 2. Essentially, each substrate carrying a terminal saturated GlcA residue except for GlcA(3-sulfate)beta1- 3GalNAc(4-sulfate)beta1-4GlcAbeta1-3GalNAc(4-sulfate) could serve as an acceptor. At a fixed substrate concentration of 28.6 µM, (GlcAbeta1-3GalNAc)(2) was the best substrate followed by (GlcAbeta1-3GalNAc)(3). The transfer rates to nonsulfated tetra-, hexa-, and octasaccharides decreased with increasing chain length. It should be noted that, as observed with the hexasaccharide serines as acceptor substrates, a 4-sulfate group on the penultimate GalNAc inhibited GalNAc incorporation onto the terminal GlcA residue. In contrast, three acceptor substrates containing a 6-sulfate group on the penultimate GalNAc residue, GlcAbeta1-3GalNAc(6-sulfate)beta1-4GlcAbeta1-3GalNAc(±4sulfate) and (GlcAbeta1-3GalNAc(6-sulfate))(3), served as good acceptors, indicating that the 6-sulfate groups have little or no inhibitory effect on chain elongation. A low but significant level of incorporation was also observed into GlcA(2-sulfate)beta1-3GalNAc(6-sulfate)beta1-4GlcAbeta1-3GalNAc(4-sulfate) despite the presence of a 2-sulfate group on the non-reducing terminal GlcA, indicating that this enzyme is not an alpha1-2-N-acetylgalactosaminyltransferase. In contrast, no incorporation was observed into the tetra- or hexasaccharide serines containing terminal unsaturated GlcA or into GlcA(3-sulfate)beta1-3GalNAc(4-sulfate)beta1-4GlcAbeta1-3GalNAc(4-sulfate) even at 143 µM. Taken together, these results strongly suggest that GalNAc was in fact transferred to the non-reducing terminal GlcA residue of the acceptor chondro-oligosaccharides in an alpha1-3 or an alpha1-4 linkage.




DISCUSSION

In this paper, we describe for the first time a unique alpha-N-acetylgalactosaminyltransferase (alpha-GalNAc T), 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 condro-oligosaccharides. Since no alpha-GalNAc-terminated structure has been so far reported in naturally occurring glycosaminoglycan chains, a question may arise whether the alpha-GalNAc T in the serum is a minor curiosity. Based upon the specific activity of the nucleotide sugar substrate, it is calculated that about 4 pmol of the alpha-GalNAc T reaction product was obtained when 1 nmol of the linkage tetrasaccharide was used as an acceptor substrate, which is comparable with about 6 pmol of the GalNAc transferase II product obtained with 300 µg (>10 nmol) of chondroitin as an acceptor substrate under the same incubation conditions described under ``Experimental Procedures.'' Thus, the alpha-GalNAc T in the serum has a comparable activity with that of the GalNAc transferase II and may play a role in galactosaminoglycan biosynthesis.

Despite the synthesis of sulfated glycosaminoglycans on the common carbohydrate-protein linkage region GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, the biosynthetic sorting mechanisms that determine whether galactosaminoglycans (chondroitin/dermatan sulfate) or glucosaminoglycans (heparin/heparan sulfate) assemble on the linkage region remain to be elucidated. We have been working on the hypothesis that there may be some structural differences in the common linkage regions among the different glycosaminoglycans. Structural studies of the linkage oligosaccharides isolated from various glycosaminoglycans revealed that the 4-sulfated and the 6-sulfated galactose residues could only be detected in the linkage region of chondroitin and/or dermatan sulfate(3, 4, 5, 6, 7, 8) . Therefore, one can suspect that the sulfate groups inhibit heparin/heparan sulfate biosynthesis and/or promote the chondroitin/dermatan sulfate pathway. In this regard, it is interesting that glycoserines and regular oligosaccharides with 4-sulfate but not 6-sulfate group(s) on the penultimate Gal or GalNAc residue showed markedly lower acceptor activity for the present novel alpha-GalNAc T (see Table 2). Although the relationship between the novel alpha-GalNAc T and chondroitin and/or dermatan sulfate biosynthesis is unclear, the observed modulation of the enzyme activity by the sulfated substrates implies that the enzyme might play an important role in the regulation of glycosaminoglycan biosynthesis.

Rohrmann et al.(21) reported that the beta-GalNAc transferase (GalNAc T-I), which catalyzes the addition of a GalNAc to the linkage tetrasaccharide, is different from that (GalNAc T-II) which catalyzes the polymerization of a chondroitin sulfate chain. This conclusion was mainly based on the differential thermostability of the two activities, i.e. GalNAc T-I was quite thermostable while GalNAc T-II was not. Interestingly, the present novel alpha-GalNAc T, which catalyzed the addition of an alpha-GalNAc to the linkage tetrasaccharide and hexasaccharide serines, was also resistant to thermodenaturation (50 °C for 60 min) as described under ``Results,'' while the serum beta-GalNAc transferase(20) , which utilized polymeric chondroitin as an acceptor substrate, underwent rapid thermodenaturation. Thus, the thermostability and substrate specificities of these two enzymes, GalNAc T-I and alpha-GalNAc T, are very similar regardless of their allegedly different linkage specificities. The discrepancy between the two enzymes regarding anomeric linkage specificity remains to be demonstrated.

Although the positional assignment of GalNAc attachment to the terminal GlcA was not accomplished in this study owing to the limited availability of authentic acceptor substrates, it seems likely that this novel enzyme may be an alpha1-4-N-acetylgalactosaminyltransferase. Very recently, Etchison et al.(24) reported that when several different cell lines were labeled with [^3H]galactose in the presence of 4-methyl umbelliferyl beta-D-xyloside (Xylbeta4MU), a small portion of the labeled products contained the carbohydrate-protein linkage region of chondroitin sulfate 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 non-reducing terminal GlcA residue in an alpha1-4 linkage(25) . Presumably, the alpha-GalNAc T discovered in the present study catalyzes the formation of a product similar to that terminating with an alpha1-4-linked GalNAc unit in cultured cells. Indeed, the nonsulfated linkage tetrasaccharide serine served as an acceptor only for the alpha-GalNAc T, despite the presence of a chondro-oligosaccharide beta-N-acetylgalactosaminyltransferase activity in fetal bovine serum. In addition, it should be noted that when the tetrasaccharide serine was tested as an acceptor using the solubilized microsomal fraction from mouse mastocytoma, it served as an acceptor only for GalNAc transferase but not for GlcNAc transferase, accepting a GalNAc residue exclusively through an alpha-linkage.^3

The role, if any, of the alpha-GalNAc T in relation to glycosaminoglycan biosynthesis is unclear. The addition of an alpha-GalNAc unit to the tetrasaccharide core of the linkage region of proteoglycans may serve as a stop signal that precludes further chain elongation. On the other hand, it may be noted that 4-epimerization of the transferred alpha-GalNAc to an alpha-GlcNAc unit may provide a primer for heparin/heparan sulfate biosynthesis ((24) ; however, see the GlcNAc transfer to GlcAbeta1-3Galbeta1-O-naphthalenemethanol described by Fritz et al.(26) ). Purification, characterization, and molecular cloning of this transferase should reveal whether it participates in the formation of glycosaminoglycan chains.


FOOTNOTES

*
This work was supported in part by the Science Research Promotion Fund from Japan Private School Promotion Foundation, Grants-in-aid for Encouragement of Young Scientists 07857169, for Scientific Research 06808061, and for Scientific Research on Priority Areas 05274107 from the Ministry of Education and Culture of Japan, the Swedish Medical Research Council Grants 2309, 10155, and 10440, and the Mizutani Foundation for Glycoscience. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-78-441-7570; Fax: 81-78-441-7571.

(^1)
The abbreviations used are: GalNAc, N-acetylgalactosamine; GlcA, D-glucuronic acid; MES, 2-(N-morpholino)ethanesulfonic acid; HPLC, high performance liquid chromatography.

(^2)
K. Sugahara, Y. Tanaka, K. Masayama, H. Kitagawa, N. Seno, and S. Yamada, manuscript in preparation.

(^3)
K. Lidholt, M. Fjelstad, U. Lindahl, F. Goto, T. Ogawa, Y. Tanaka, K. Tsuchida, H. Kitagawa, and K. Sugahara, manuscript in preparation.


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