©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of Heparan Sulfate 6-Sulfotransferase from the Culture Medium of Chinese Hamster Ovary Cells (*)

(Received for publication, September 9, 1994; and in revised form, November 28, 1994)

Hiroko Habuchi Osami Habuchi (1) Koji Kimata (§)

From the Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11 Department of Life Science, Aichi University of Education, Kariya 448, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Heparan sulfate 6-sulfotransferase, which catalyzes the transfer of sulfate from 3`-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified 10,700-fold to apparent homogeneity with a 40% yield from the serum-free culture medium of Chinese hamster ovary cells. The isolation procedure included affinity chromatography of the first heparin-Sepharose CL-6B column (stepwise elution), 3`,5`-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradient elution). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 52 and 45 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the amino group of glucosamine residue or to position 2 of the iduronic acid residue. Heparan sulfate was also sulfated by the purified enzyme at position 6 of N-sulfoglucosamine residue. Chondroitin and chondroitin sulfate did not serve as acceptors. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated strongly by protamine. The K value for adenosine 3`-phosphate 5`-phosphosulfate was 0.44 µM.


INTRODUCTION

Heparan sulfate and heparin interact with a variety of proteins, such as growth factors, extracellular matrix components, and protease inhibitors, suggesting their involvement not only in a variety of cellular aspects, such as cell growth, differentiation, and cell adhesion, but also in the anticoagulation process and some pathological processes, such as viral infections(1, 2, 3) . Interactions of heparan sulfate/heparin with those ligands seem to be mediated by the binding of ligands to specific structures in heparan sulfate/heparin. For example, the basic fibroblast growth factor interacts with a cluster of GlcNSO(3)-IdoA(2SO(4)) in heparan sulfate (4, 5, 6, 7, 8) , (^1)and its high affinity receptor appears to interact with some specific sites containing GlcNSO(3)(6SO(4))-IdoA(2SO(4)) in heparan sulfate(8) . It has recently been suggested that heparan sulfate chains of cell surface and extracellular matrix heparan sulfate proteoglycan, which could be distinguished from any known proteoglycan such as syndecan, fibroglycan, or glypican, regulate basic fibroblast growth factor receptor binding and thereby regulate the biological activity of basic fibroblast growth factor(9) . The response of neural cells to either acidic or basic fibroblast growth factor appears to be regulated by developmentally modulated forms of heparan sulfate proteoglycans (10) . Heparan sulfate from highly metastatic tumor cells exhibited a higher degree of sulfation than that from low metastatic tumor cells, which was due to increased contents of 6-O-sulfated glucosamine residue(11) . Microheterogeneity in the heparan sulfate structure, particularly in sulfation patterns at various positions, may play an important role in these cellular aspects. Thus, it is important to study how the microheterogeneity is yielded and regulated. It is suggested that O-sulfation is the final step in the modification of the structure during the biosynthesis of heparin and probably heparan sulfate as well(12, 13) . Therefore, O-sulfation at various positions of heparan sulfate is an important step in determining the structure of each functional domain in heparan sulfate. Various types of sulfotransferases have been shown to be responsible for the sulfation of heparin and heparan sulfate: sulfation of 2-N(14, 15, 16, 17) , 6-O(16, 19) , and 3-O(20) of glucosamine residue, sulfation of 2-O(19) of L-iduronic acid residue, and sulfation of 2-O(21) of D-glucuronic acid residue (see Fig. 1). However, only N-sulfotransferases have been purified to homogeneity from rat liver and mouse mastocytoma(14, 15) . More recently, molecular cloning studies have suggested that these N-sulfotransferases were closely related but were clearly distinct from each other(22, 23, 24, 25) , suggesting that the biosyntheses of heparan sulfate and heparin may be catalyzed by different enzymes and independently regulated.


Figure 1: Partial structures of heparan sulfate with possible sulfation positions and the sulfation sites by heparan sulfate 6-sulfotransferase. Arrows indicate the sulfation sites by purified heparan sulfate 6-sulfotransferase. It remains to be determined whether or not purified heparan sulfate 6-sulfotransferase catalyzes the transfer of sulfate to position 6 of GlcNSO(3) adjacent to GlcA (broken arrow).



We recently purified chondroitin 6-sulfotransferase with a high yield from the serum-free culture medium of chick chondrocytes(26) . We have found in the present study that heparan sulfate 6-sulfotransferase (Fig. 1) was likewise secreted into the serum-free culture medium of CHO cells, although the amount of secreted heparan sulfate 6-sulfotransferase was only one-fiftieth of the amount of chondroitin 6-sulfotransferase secreted from the chondrocytes. In this paper, we describe the purification to apparent homogeneity and some properties of heparan sulfate 6-sulfotransferase from the serum-free culture medium of CHO cells.


EXPERIMENTAL PROCEDURES

Materials

[S]H(2)SO(4) was purchased from the Japan Radioisotope Association (Tokyo). Dulbecco's modified Eagle's medium, trypsin (from bovine pancreas, type III), unlabeled PAPS, 3`,5`-ADP-agarose, and heparin were from Sigma. Fetal bovine serum was from Cytosystems PTY Ltd. Fast desalting column HR10/10, heparin-Sepharose CL-6B, and Superose 12 column HR10/30 were from Pharmacia Biotech Inc. (Uppsala, Sweden). Partisil-10 SAX column was from Whatman (Clifton, NJ), PAMN column was from YMC (Kyoto, Japan), and Chondroitinase ABC, heparitinase I, heparitinase II, heparitinase III, chondroitin sulfate A (whale cartilage, 4-sulfate unit, 6-sulfate unit (80:20)), heparan sulfate (pig aorta), completely desulfated and N-resulfated heparin (CDSNS-heparin), N-desulfated heparin, and unsaturated glycosaminoglycan disaccharide kit were from Seikagaku Corporation (Tokyo). Recombinant N-glycanase was from Genzyme Co. [S]PAPS was prepared as described previously(27) . Chondroitin (squid skin) was prepared as described previously(28) .

Cell Culture and Collection of Medium

CHO cells were inoculated in roller bottles (In Vitro Science Products, Inc.) at a density of about 3.3 times 10^7 cells/bottle and cultured for 2 days in 100 ml of Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum supplemented with 10 mM Hepes pH 7.2 buffer, penicillin, and streptomycin. Then the culture medium was replaced every other day with 100 ml of Cosmedium-001 supplemented with 50 µg/ml ascorbic acid and 10 mM Hepes buffer. The culture in Cosmedium-001 was continued for 10 days. The spent medium was pooled and centrifuged at 1,000 times g for 5 min. To the supernatant solution, Tris-HCl, pH 7.2 (10 mM), Triton X-100 (0.1% (w/v)), MgCl(2) (10 mM), CaCl(2) (2 mM), and glycerol (20% (v/v)) were added to the final concentrations indicated in parentheses. The pooled medium containing these additives (16 liters) was designated as the buffered medium fraction and stored at -20 °C until used.

Purification of Heparan Sulfate 6-Sulfotransferase Secreted into the Medium

All operations were performed at 4 °C.

Step 1: First Chromatography on Heparin-Sepharose CL-6B

0.1 volume of the buffered medium fraction was applied to a column of heparin-Sepharose CL-6B (20 times 65 mm, 20 ml) equilibrated with 0.15 M NaCl in buffer A (10 mM Tris-HCl, pH 7.2, 0.1% (w/v) Triton X-100, 10 mM MgCl(2), 2 mM CaCl(2), 20% (v/v) glycerol) at the flow rate of 70 ml/h. The column was washed with 200 ml (10 volumes of a column) and then eluted with 100 ml (5 volumes of a column) of 1.0 M NaCl in buffer A. The chromatography was repeated 10 times. The eluates from 10 columns were combined, concentrated to 100 ml with polyethylene glycol 20,000 (molecular weight 15,000-25,000), and dialyzed against 0.05 M NaCl in buffer A.

Step 2: Chromatography on 3`,5`-ADP-agarose

Half of the fraction from step 1 was applied to a column of 3`,5`-ADP-agarose (14 times 90 mm, 1.9 µmol of 3`,5`-ADP/ml of gel) equilibrated with buffer A containing 0.05 M NaCl at a flow rate of 13 ml/h. The column was washed with 120 ml of buffer A containing 0.05 M NaCl. The sulfotransferase activity was eluted with a linear gradient of 0-0.2 mM 3`,5`-ADP in buffer A containing 0.05 M NaCl of total volume (150 ml). The fractions containing sulfotransferase activity (indicated by a horizontal bar in Fig. 3) were pooled. The chromatography was performed twice, and the fractions containing the activity were combined.


Figure 3: 3`,5`-ADP-agarose affinity chromatography of the first heparin-Sepharose fractions. The fractions eluted from the heparin-Sepharose column with buffer A containing 1 M NaCl were concentrated, dialyzed exhaustively against 0.05 M NaCl in buffer A, and applied to a 3`,5`-ADP-agarose column as described under ``Experimental Procedures.'' Fractions of 2 ml were collected. Heparan sulfate O-sulfotransferase activity (bullet) and protein concentration (circle) of each fraction were assayed. The broken line indicates the concentration of 3`,5`-ADP. The arrow indicates the elution with buffer A containing 1 M NaCl. The horizontal bar indicates the fractions that were pooled for further purification.



Step 3: Second Chromatography on Heparin-Sepharose CL-6B

The step 2 fraction was applied to a heparin-Sepharose CL-6B column (16 times 35 mm, 5 ml) equilibrated with buffer A containing 0.15 M NaCl at the flow rate of 13 ml/h. The column was washed with 50 ml of buffer A containing 0.25 M NaCl. The sulfotransferase activity was eluted with a linear gradient from 0.25 to 1.2 M NaCl in buffer A (total volume, 150 ml). The fractions containing sulfotransferase activity were pooled (indicated by a horizontal bar in Fig. 4A) and dialyzed against 0.15 M NaCl in buffer A. The fraction was stored at -20 °C.


Figure 4: Second heparin-Sepharose CL-6B chromatography of the 3`,5`-ADP-agarose fraction. A, the sulfotransferase fraction from 3`,5`-ADP-agarose (indicated by horizontal bar in Fig. 3) was applied to a heparin-Sepharose column as described under ``Experimental Procedures.'' Fractions of 2 ml were collected. After washing with buffer A containing 0.25 M NaCl, the column was eluted with a linear gradient of NaCl. Chondroitin sulfotransferase activity (circle) and heparan sulfate O-sulfotransferase activity (bullet) of each fraction were assayed. The broken line indicates the concentration of NaCl. Protein concentration was not determined because of very low content. B, aliquots of every three fractions that showed the activity were analyzed by SDS-PAGE (10% gel). Proteins were visualized with silver nitrate stain. Molecular size standards were phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (20.1 kDa). The sharp band at 55 kDa in the fractions is likely due to the artifact of silver staining because this sharp band did not appear reproducibly.



Extraction of Sulfotransferases from the Cultured CHO Cells

For extraction of sulfotransferase in the cells, the cell layer was washed with phosphate-buffered saline, scraped off the dish in 5 ml of buffer A containing 0.15 M NaCl, and homogenized in a glass homogenizer. The concentration of Triton X-100 in the homogenate was then increased to 0.5%. After 1 h of gentle stirring, the homogenate was centrifuged at 4 °C for 10 min at 10,000 times g. The supernatant was applied to a small heparin-Sepharose CL-6B column (bed volume, 0.6 ml), and washed with 5 ml of 0.15 M NaCl in buffer A, and then eluted with 5 ml of 1.0 M NaCl in buffer A.

Assay for Sulfotransferase Activity

The standard reaction mixture contained 2.5 µmol of imidazole HCl, pH 6.8, 3.75 µg of protamine chloride, 25 nmol (as hexosamine) of CDSNS-heparin, 50 pmol of [S]PAPS (about 5 times 10^5 cpm), and enzyme in a final volume of 50 µl. The reaction mixtures were incubated at 37 °C for 20 min, and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. 0.1 µmol (as glucuronic acid) of chondroitin sulfate A was added to the reaction mixture as a carrier. S-Labeled polysaccharides were precipitated with 3 volumes of cold ethanol containing 1.3% potassium acetate and separated completely from [S]PAPS and its degradation products by gel chromatography using Fast desalting columns as described previously(26) . The incorporation of [S] was linear with the amounts of proteins and with the incubation time under the above conditions (up to 5.8 ng of protein and up to 30 min when the purified enzyme was used) (data not shown). One unit of enzyme activity was defined as the amount required to transfer 1 pmol of sulfate/min.

SDS-Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gel electrophoresis of proteins in SDS was carried out on a 10% polyacrylamide gel as described previously(29) . Protein bands were detected by silver staining or with Coomassie Brilliant Blue.

Detection of Sulfotransferase Activity in Gels after SDS-PAGE

Extraction and renaturation of enzyme from the gels after SDS-PAGE were carried out essentially as described by Lind et al.(30) as for heparin D-glucuronosyltransferase and the N-acetyl-D-glucosaminyltransferase. Before electrophoresis, the final purified enzyme in buffer A containing 0.15 M NaCl was heated at 50 °C for 30 min in the SDS-PAGE sample buffer. The gel was washed with 0.05 M Tris-HCl, pH 8.0, and was cut into 3- or 5-mm segments and subjected to agitation at 4 °C with 100 µl of buffer A containing 0.15 M NaCl for 48 h. The eluted enzyme was collected by centrifugation and assayed for the sulfotransferase activity.

Gel Chromatography of Sulfotransferase on Superose 12

Superose 12 HR 10/30 was equilibrated with buffer A containing 2 M NaCl. 200 µl of sample was injected and eluted with buffer A containing 2 M NaCl at a flow rate of 0.25 ml/min.

Identification of Enzymatic Reaction Products

S-labeled glycosaminoglycans were digested with a mixture of 5 milliunits of heparitinase I, 0.5 milliunit of heparitinase II, and 5 milliunits of heparitinase III in 50 µl of 50 mM Tris-HCl, pH 7.2, 1 mM CaCl(2), 2 µg of bovine serum albumin at 37 °C for 2 h. The specificity of each heparitinase is as follows. Heparitinase I is specific for GlcNAc or GlcNSO(3) residues adjacent to GlcA in heparan sulfate. Heparitinase II is also specific for the GlcNAc and GlcNSO(3) residues but not for GlcNSO(3)(3SO(4)) residue in heparan sulfate/heparin. Heparitinase III is specific for the GlcNSO(3)(6SO(4)) residues adjacent to IdoA(2SO(4)) in heparan sulfate/heparin (see (31) for the details). The digested products were injected together with standard unsaturated disaccharides in a column of PAMN (4.6 mm times 25 cm). The column was developed by HPLC as described previously except for a flow rate of 1.2 ml/min(4) . 0.6-ml fractions were collected and mixed with 3 ml of Ready Safe Scintillator, and the radioactivity was determined. Degradation of the S-labeled glycosaminoglycans with nitrous acid at pH 1.5 and reduction of the product with NaBH(4) were carried out as described by Shively and Conrad (32) . Aliquots of the products were subjected to paper electrophoresis, and the paper strips were cut into 1.25-cm segments and counted. Released radioactive free sulfate represents the sulfate bound to the amino group of the glucosamine. Remainders of the products were subjected to gel filtration connecting two Fast desalting columns that had been equilibrated with 0.5 M NH(4)HCO(3). The fractions corresponding to S-labeled disaccharides were collected and analyzed by HPLC on a Partisil-10 SAX column as described previously(4, 33) .

Other Methods

The galactosamine and glucosamine contents of glycosaminoglycans were determined by the Elson-Morgan method as modified by Strominger et al.(34) after hydrolysis of the glycosaminoglycans with 6 M HCl at 100 °C for 4 h. Protein concentration was determined by a BCA kit (Pierce) using bovine serum albumin as a standard. 0.5 µg of the enzyme protein was digested with 0.5 unit of N-glycanase at 37 °C for 16 h according to the method recommended by the manufacturer except for omitting mercaptoethanol.


RESULTS

Secretion of Heparan Sulfate 6-Sulfotransferase into the Serum-free Medium

Sulfotransferase activity was secreted into the serum-free medium (Cosmedium-001 supplemented with sodium ascorbate) under the conditions used (Table 1). The spent medium of CHO cells contained the highest sulfotransferase activity among the three cell lines tested. S-Labeled polysaccharides, which were formed from CDSNS-heparin by incubation with the culture medium of CHO cells or FM3A cells, were digested with heparitinase. The products were identified by HPLC as described under ``Experimental Procedures.'' Major radioactivity was recovered in a peak of DeltaDi-(N,6)diS, and a small amount of radioactivity was detected in a peak of DeltaDi-(N,U)diS as a shoulder (peaks 3 and 4, respectively, in Fig. 2, A and B). The ratio of DeltaDi-(N,U)diS to DeltaDi-(N,6)diS was 0.04 for CHO cells and 0.25 for FM3A cells. No radioactive sulfate was released when S-labeled products were subjected to nitrous acid degradation at pH 1.5 (Fig. 2C). The results indicate that only O-sulfation occurred in the substrate without any N-sulfation under the reaction conditions used. Furthermore, the results also indicate that major sulfotransferase secreted by CHO cells catalyzed the transfer of sulfate to position 6 of N-sulfoglucosamine residue in heparan sulfate (this enzyme was hereafter designated as heparan sulfate 6-sulfotransferase) (Fig. 1). The activity of heparan sulfate 6-sulfotransferase in the CHO cell layer extract was 0.65 unit/2 times 10^7 cells and less than 10% of the activity in the culture medium.




Figure 2: Analysis of S-labeled products derived from the incubation of CDSNS-heparin, [S]PAPS, and the enzymes. The enzymes were prepared from the spend medium of CHO cells (A and C), and FM3A cells (B and D), respectively, as described in Table 1. The sulfotransferase reaction, digestion of the product with a mixture of heparitinase, and subsequent HPLC of the digested products on a polyamine-bound silica PAMN column were carried out as described under ``Experimental Procedures'' (A and B). Broken lines indicate a gradient concentration of KH(2)PO(4). Arrows indicate the elution positions of: DeltaDi-6S (1), DeltaDi-NS (2), DeltaDi-(N,6)diS (3), DeltaDi-(N,U) diS (4), DeltaDi-(N,6,U)triS (5). Products of the degradation with nitrous acid at pH 1.5 were subjected to paper electrophoresis as described under ``Experimental Procedures'' (C and D). The dotted lines indicate the migration of S-free sulfate as standard.



Purification of Heparan Sulfate 6-Sulfotransferase

Based on the above observations, we decided to use the spent medium of CHO cells as an enzyme source and succeeded in purifying the enzyme to an apparent homogeneity with about 10,700-fold purification as described under ``Experimental Procedures.'' Table 2shows a summary of the purification of the sulfotransferase from 16 liters of the buffered medium fraction. The details for each step are as follows.



Step 1: First Heparin-Sepharose Chromatography

The buffered medium fraction was applied to a heparin-Sepharose column equilibrated with buffer A containing 0.15 M NaCl. Most proteins absorbed to the column were eluted in 0.25 M NaCl fraction, and heparan sulfate 6-sulfotransferase activity was eluted in 1 M NaCl fraction. Compared with chondroitin 6-sulfotransferase(26) , heparan sulfate 6-sulfotransferase had a high affinity to heparin. After this chromatography, the total activity of heparan sulfate 6-sulfotransferase increased about 1.6-fold, suggesting that some inhibitors of heparan sulfate 6-sulfotransferase and/or degradating enzymes of PAPS might be removed at this purification step.

Step 2: 3`,5`-ADP-agarose Chromatography

This column chromatography resulted in a 35-fold purification of the sulfotransferase (Fig. 3). Because the sulfotransferase appeared to be less stable in 0.05 M NaCl than in 0.15 M NaCl, each eluate was collected in tubes containing 1.0 M NaCl in buffer A to make the solution of 0.15 M NaCl at the final concentration. 3`,5`-ADP strongly inhibited heparan sulfate 6-sulfotransferase as described previously in other glycosaminoglycan sulfotransferases(15, 26) . The concentration of 3`,5`-ADP giving a 50% inhibition was less than 2.5 µM. Enzyme activity at this step, therefore, was determined after the removal of 3`,5`-ADP by absorbing the enzyme to a small heparin-Sepharose column and eluting with 1 M NaCl in buffer A.

Step 3: Second Heparin-Sepharose Chromatography

Chondroitin sulfotransferase activity was eluted at the lower NaCl concentration than heparan sulfate 6-sulfotransferase activity (Fig. 4A). This chondroitin sulfotransferase activity catalyzed the transfer of sulfate to position 4 of N-acetylgalactosamine residue in chondroitin or chondroitin sulfate A but not to position 6 of N-acetylgalactosamine residue (data not shown). Fig. 4B shows SDS-PAGE for fraction numbers 25-49 in the second heparin-Sepharose column chromatography. Judging from the elution pattern of the enzyme activity, two protein bands of M(r) 52,000 and 45,000 seem to correspond to the sulfotransferase.

Purity of the Heparan Sulfate 6-Sulfotransferase

Samples at each purification step were analyzed by SDS-PAGE (Fig. 5). Two bands of M(r) 52,000 and 45,000 were predominantly stained with silver nitrate in the second heparin-Sepharose fraction (Fig. 5, lane 4). There was no significant difference in the mobilities of the two protein bands before and after reduction (Fig. 5, lane 4 versus lane 5). In order to see if the two protein bands were responsible for the enzyme activity, proteins in the gel segments following SDS-PAGE of the purified enzyme were extracted and assayed for heparan sulfate 6-sulfotransferase activity after renaturation as described under ``Experimental Procedures.'' Extracts from the segments corresponding to the 52- and the 45-kDa protein bands showed significant activity with a ratio of 1 to 2 (varied between experiments), irrespective of the low recovery of the activity (less than 2%) (data not shown). When the second heparin-Sepharose fraction was applied to a Superose 12 column equilibrated with buffer A containing 2 M NaCl, heparan sulfate 6-sulfotransferase activity was eluted in the fractions around M(r) 49,000 (Fig. 6), which was between the molecular weights of the two protein bands on SDS-PAGE. These observations suggested that both protein bands bear the sulfotransferase activity. When the N-glycanase digest of this fraction was subjected to SDS-PAGE, protein bands of 52 and 45 kDa disappeared, whereas protein bands of 43 and 38 kDa appeared (Fig. 7). This result indicated that both proteins were glycoproteins containing more than 15% of carbohydrate.


Figure 5: SDS-PAGE of heparan sulfate 6-sulfotransferase fractions at various purification steps. Lane 1, 0.6 µg of protein of the buffered medium fraction; lane 2, 0.6 µg of protein eluted with 1 M NaCl in buffer A from the first heparin-Sepharose CL-6B column; lane 3, 0.6 µg of protein eluted with a 3`,5`-ADP gradient from 3`,5`-ADP-agarose column; lane 4, 0.15 µg of protein eluted with a NaCl gradient from the second heparin-Sepharose CL-6B column; lane 5, 14 µg of protein from the same fraction as in lane 4, except the sample in lane 5 was reduced only with 5% mercaptoethanol before electrophoresis. Lanes 1-4 were stained with silver nitrate. Lane 5 was stained with Coomassie Brilliant Blue. Molecular size standards were the same as in Fig. 4B.




Figure 6: Superose 12 gel chromatography of heparan sulfate 6-sulfotransferase. Heparan sulfate 6-sulfotransferase eluted from the second heparin-Sepharose CL-6B column was concentrated and dialyzed against buffer A containing 2 M NaCl. 200 µl of concentrated solution was injected into a Superose 12 column and eluted with buffer A containing 2 M NaCl as described under ``Experimental Procedures.'' Heparan sulfate 6-sulfotransferase activity (bullet) of each fraction was assayed. The arrows indicate the elution positions of bovine serum albumin (68 kDa) (1), ovalbumin (45 kDa) (2), and chymotrypsinogen A (25 kDa) (3).




Figure 7: SDS-PAGE of heparan sulfate 6-sulfotransferase treated with N-glycanase. Lane 1, 0.15 µg of the purified enzyme protein; lane 2, digests of 0.15 µg of the purified enzyme protein with N-glycanase; lane 3, the same amount of N-glycanase as in lane 2. Proteins were stained with silver nitrate. Molecular size standards were the same as in Fig. 4B.



Specificity for Acceptor Substrates of Heparan Sulfate 6-Sulfotransferase

The purified fraction of heparan sulfate 6-sulfotransferase (after step 3) was incubated with different acceptors. The purified sulfotransferase was able to transfer sulfate to CDSNS-heparin and heparan sulfate (from pig aorta) (Table 3). However, N-desulfated heparin was a poor acceptor. The enzyme showed no activity toward chondroitin and chondroitin sulfate A. To determine the position of the sulfate group transferred to CDSNS-heparin, S-labeled product derived from CDSNS-heparin was digested with a mixture of heparitinase I, II, and III, and the products were then subjected to HPLC (Fig. 8A). Most of radioactivity was eluted at the position of DeltaDi-(N,6)diS, and only slight radioactivity was detected at the position of DeltaDi-(N,6,U)triS. Furthermore, HPLC of S-labeled disaccharides formed by nitrous acid degradation at pH 1.5 showed that most of the radioactivity was eluted at the position of IdoA-AMan(R)(6SO(4)) (Fig. 8B). S-Free sulfate was not detected in the nitrous acid degradation product. These observations showed that the purified enzyme catalyzed sulfation of position 6 of the N-sulfoglucosamine residue of IdoA-GlcNSO(3) unit in CDSNS-heparin. In contrast, when heparan sulfate (from pig aorta) was used as acceptor, heparitinase digestion yielded peaks corresponding to DeltaDi-(N,6)diS and DeltaDi-(N,6,U)triS with approximately equal radioactivity and unidentified peaks with only small radioactivity (retention time, 19 and 36.5 min) (Fig. 9).




Figure 8: HPLC on PAMN column of S-labeled CDSNS-heparin produced by incubation with [S]PAPS and the purified heparan sulfate 6-sulfotransferase (A) and HPLC on Partisil-10 SAX column of the products of degradation with nitrous acid at pH 1.5 (B). A, the products of sulfotransferase reaction were digested with a mixture of heparitinases and subjected to PAMN column as described under ``Experimental Procedures.'' The broken line indicates the concentration of KH(2)PO(4). The arrows indicate the same as in Fig. 2. B, the products of nitrous acid degradation at pH 1.5 were subjected to gel filtration, and the disaccharide fraction was applied to a Partisil-10 SAX column. The conditions of HPLC were as described under ``Experimental Procedures.'' The broken line indicates the concentration of KH(2)PO(4). The arrows indicate the elution position of HexA-AMan(R) (1), GlcA(2SO(4))-AMan(R) (2), GlcA-AMan(R)(6SO(4)) (3), IdoA-AMan(R)(6SO(4)) (4), IdoA(2SO(4))-AMan(R) (5), and IdoA(2SO(4))-AMan(R)(6SO(4)) (6).




Figure 9: Analysis of S-labeled heparan sulfate produced by incubation with [S]PAPS and the purified heparan sulfate 6-sulfotransferase. After the sulfotransferase reaction, the samples were digested with a mixture of heparitinases and applied to HPLC as described under ``Experimental Procedures.'' The conditions of HPLC on a polyamine-bound silica PAMN column were as described under ``Experimental Procedures.'' The broken line indicates the concentration of KH(2)PO(4). The arrows are the same as indicated in Fig. 2.



Properties of Heparan Sulfate 6-Sulfotransferase

The pH dependence of heparan sulfate 6-sulfotransferase activity was shown in Fig. 10A. The maximum activity was observed at pH 6.2-6.4. Dithiothreitol (DTT) decreased the enzyme activity to 42 and 19% of control in 2 and 10 mM DTT, respectively (Fig. 10B). NaCl stimulated the enzyme activity (Fig. 10C). The maximum activity was observed around 175 mM NaCl in the absence of DTT and around 100 mM NaCl in the presence of 2 mM DTT. The effects of NaCl on heparan sulfate 6-sulfotransferase were quite different from those on N-sulfotransferase. In case of the latter enzyme, NaCl decreased the activity. Protamine activated heparan sulfate 6-sulfotransferase remarkably as observed with chondroitin 4- and 6-sulfotransferase (Fig. 10D). The K(m) value of heparan sulfate 6-sulfotransferase for PAPS was 4.4 times 10M (Fig. 11).


Figure 10: Properties of the purified heparan sulfate 6-sulfotransferase. A, pH dependence of heparan sulfate 6-sulfotransferase activity. The sulfotransferase activities were determined as described under ``Experimental Procedures,'' except that the pH was varied using 2.5 µmol of Tris-HCl (circle), 2.5 µmol of imidazole HCl (bullet), 2.5 µmol of Mes (box), or 2.5 µmol of potassium acetate () buffer. B, effect of DTT on the sulfotransferase activity. The reaction mixtures contained various amounts of DTT. C, effect of NaCl on the sulfotransferase activity. The reaction mixture contained various concentrations of NaCl with (circle) or without (bullet) 2 mM DTT. D, effect of protamine on the sulfotransferase activity. The reaction mixture contained various amounts of protamine.




Figure 11: K value of heparan sulfate 6-sulfotransferase for PAPS. The sulfotransferase activity was determined using 2.12 ng of the purified enzyme as described under ``Experimental Procedures'' except that various amounts of PAPS were added. The inset shows the reciprocal plot.




DISCUSSION

In our present experiments, heparan sulfate 6-sulfotransferase was purified to an apparent homogeneous level from the culture medium of CHO cells. Chondroitin 6-sulfotransferase was also purified from the culture medium of chondrocytes. Although the mechanisms of the secretion of these sulfotransferases are not clear, the results may suggest that conditioned media may be useful sources for the purification of glycosaminoglycan sulfotransferases. As was also the case with heparan sulfate/heparin N-sulfotransferase and chondroitin 6-sulfotransferase(14, 15, 26) , affinity chromatography with heparin-Sepharose and 3`,5`-ADP-agarose yielded successful purification of heparan sulfate 6-sulfotransferase.

When CDSNS-heparin was used as an acceptor, the purified sulfotransferase was found to transfer sulfate exclusively to position 6 of N-sulfoglucosamine residue and not to transfer sulfate to position 2 of hexuronic acid residue or position 2 of glucosamine residue. The evidences for this conclusion are that: 1) digestion of S-labeled product derived from CDSNS-heparin with a heparitinase mixture yielded DeltaDi-(N,6)diS as a major S-labeled disaccharide product, 2) degradation with nitrous acid at pH 1.5 yielded IdoA-AMan(6SO(4)) as a major S-labeled disaccharide product, and 3) no free SO(4) was released when the S-labeled product derived from CDSNS-heparin was treated with nitrous acid at pH 1.5. Wlad et al. (35) have recently reported that both the 6-sulfotransferase and the 2-sulfotransferase from mouse mastocytoma may be part of the same protein. The observed difference of the enzyme specificity between their and our sulfotransferases might be due to the following reasons. Because our enzyme was obtained from culture medium, the 6-sulfotransferase might have been produced as a secretary protein by protease processing from the single protein that contained both O-sulfotransferase activities. Because these enzymes were produced by different types of cells, they might be different gene products. We prepared the 6-sulfotransferase from CHO cells, which are engaged in the biosynthesis of heparan sulfate. On the other hand, Wald et al.(35) prepared the enzyme from mouse mastocytoma tissue, which is mainly involved in the biosynthesis of heparin. As with N-sulfotransferases(22, 23, 24, 25) , it is possible that O-sulfation of heparin and heparan sulfate may be catalyzed by different enzymes.

When heparan sulfate was used as acceptor for the purified enzyme, digestion of the S-labeled product with the heparitinase mixture yielded nearly equal amounts of DeltaDi-(N,6)diS and DeltaDi-(N,6,U)triS. Because the purified sulfotransferase preparation was unable to transfer sulfate to position 2 of the hexuronic acid residue of the HexA-GlcNSO(3) unit in CDSNS-heparin, it is possible that the S-labeled HexA(2SO(4))-GlcNSO(3)(6SO(4)) unit in the S-heparan sulfate product was formed by the sulfation of position 6 of N-sulfoglucosamine residue in the HexA(2SO(4))-GlcNSO(3) unit. The content of the HexA(2SO(4))-GlcNSO(3) unit was 4.4 and 0.2% in heparan sulfate (pig aorta) and CDSNS-heparin, respectively. The relatively higher content of the HexA(2SO(4))-GlcNSO(3) unit in the heparan sulfate may have caused the higher production of the S-labeled HexA(2SO(4))-GlcNSO(3)(6SO(4)) unit. However, the possibility cannot be completely ruled out that the HexA(2SO(4))-GlcNSO(3)(6SO(4)) unit was formed by the transfer of sulfate to position 2 of hexuronic acid residue of the HexA-GlcNSO(3)(6SO(4)) unit. If heparan sulfate 6-sulfotransferase is able to catalyze the transfer of sulfate not only to position 6 of N-sulfoglucosamine residue in the HexA-GlcNSO(3) unit but also to position 6 of N-sulfoglucosamine residue in the HexA(2SO(4))-GlcNSO(3) unit, this specificity of the enzyme could be consistent with the previous observations that the IdoA(2SO(4))-GlcNSO(3)(6SO(4)) unit was formed by the sulfation of position 6 of GlcNSO(3) residue after or simultaneously with the sulfation of position 2 of the IdoA residue(18) . Kusche et al., using various pentasaccharides GlcNSO(3)-GlcA/IdoA-GlcNSO(3)-GlcA/IdoA-GlcNSO(3) as acceptors and mouse mastocytoma microsome as an enzyme, also reported that sulfation at the position 6 of the internal glucosamine unit took place, irrespective of the structure of the adjacent hexuronic acid residue(19) . The purified heparan sulfate 6-sulfotransferase can catalyze the transfer of sulfate at least to position 6 of GlcNSO(3) adjacent to the iduronic acid unit. However, it remains to be determined whether or not the purified heparan sulfate 6-sulfotransferase catalyzes the transfer of sulfate to position 6 of GlcNSO(3) adjacent to GlcA. It has also been noticed that, when heparan sulfate was used as an acceptor, the S-labeled GlcA-GlcNAc(6SO(4)) unit was not detected in the products, although the acceptor contained 64% of the GlcA-GlcNAc unit (Fig. 9). The result suggests that the C-6 sulfation of GlcNAc in heparan sulfate may be performed by a different sulfotransferase.

It is interesting to compare the properties of heparan sulfate 6-sulfotransferase with those of other purified glycosaminoglycan sulfotransferase. DTT inhibited heparan sulfate 6-sulfotransferase but not chondroitin 6-sulfotransferase(36) . On the contrary, DTT stimulated chondroitin 4-sulfotransferase(36) .

Heparan sulfate 6-sulfotransferase appears to be a monomer protein, because the molecular weight of the purified sulfotransferase determined by SDS-PAGE was consistent with that determined by Superose 12 gel chromatography (Fig. 6). Rat liver heparan sulfate N-deacetylase/N-sulfotransferase is a monomer (14, 37) and chick chondrocyte chondroitin 6-sulfotransferase may be a dimer. A majority of proteins intrinsic to the Golgi apparatus membrane appear to be dimers in situ(38) . The apparent K(m) value for PAPS of the purified heparan sulfate 6-sulfotransferase was 4.4 times 10M, whereas that of heparan sulfate N-sulfotransferase of rat liver was 1.08 times 10M(39) . Heparan sulfate 6-sulfotransferase, therefore, appears to have higher affinity to PAPS than heparan sulfate N-sulfotransferase. The difference in the value of K(m) for PAPS between heparan sulfate 6-sulfotransferase and heparan sulfate N-sulfotransferase, however, might be due to the difference in the assay conditions used. For example, as a cationic activator, protamine was added to the present assay mixture for heparan sulfate 6-sulfotransferase, whereas Mg and Mn were added to the reported assay mixture for heparan sulfate N-sulfotransferase. We previously observed that cationic proteins such as protamine and histone stimulated chondroitin 6-sulfotransferase by decreasing the K(m) value for PAPS(28) . The low K(m) value for PAPS of heparan sulfate 6-sulfotransferase may have been caused by the presence of protamine.

SDS-PAGE of the purified enzyme fraction gave two protein bands of 52 and 45 kDa. Both protein bands were always comigrated in the gel when the peak fractions containing the activity of heparan sulfate 6-sulfotransferase from heparin-Sepharose or Superose 12 chromatography were subjected to SDS-PAGE. Both protein bands decreased their molecular masses by treatment with N-glycanase, suggesting that the difference in the molecular masses of the two proteins may not be attributed solely to the difference in the content of N-glycosidic carbohydrate chains. When proteins were extracted from the gels following SDS-PAGE and assayed for 6-sulfotransferase after renaturation, the significant activity of 6-sulfotransferease was recovered from the gels containing these two protein bands. The results suggested that both the 52- and 45-kDa proteins bear 6-sulfotransferase activity. Our preliminary studies on the amino-terminal amino acid sequences of the two proteins showed that at least 11 amino acid residues were completely identical, (^2)which suggested a close relationship between these two proteins. Several glycosyltransferases that are derived from the single genes exhibit catalytically active multiple forms with different molecular weights(40, 41, 42, 43) . This size difference has been supposed to be due to limited proteolytic cleavage, binding of detergents or lipids, or aggregation. In addition, there are some reports suggesting that the stem regions are cleaved off proteolytically when proteins originally present in the Golgi apparatus, such as glycosyltransferases, were secreted(43, 44) . For now, it remains to be studied whether these two proteins of heparan sulfate 6-sulfotransferase are closely related proteins or distinct ones.

Heparan sulfate prepared from CHO cells contains HexA(2SO(4)) residue and GlcNSO(3)(6SO(4)) residue in a proportion of 4:3. However, IdoA 2-O-sulfotransferase activity was only 4% of total heparan sulfate O-sulfotransferase activity in the culture medium of CHO cells. It is not certain why IdoA 2-O-sulfotransferase activity was hardly detected in the cultured medium of CHO cells. The following are possible reasons to explain why scarce activity of IdoA 2-O-sulfotransferase was detected in the culture medium: 1) the processing of IdoA 2-O-sulfotransferase to soluble forms may not be as efficient as the processing of heparan sulfate 6-sulfotransferase; 2) IdoA 2-O-sulfotransferase may need cofactors for its enzyme activity that were not secreted into the culture medium; or 3) there may be no relationship between the enzyme activity and the amount of the product. These possibilities remain to be studied.


FOOTNOTES

*
This work was supported by grants-in-aid from the Ministry of Education, Culture, and Science, Japan, by the Special Coordination Funds of the Science and Technology Agency of the Japanese Government, and by a special research fund from Seikagaku Corporation. 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 all correspondence should be addressed. Tel.: 81-52-264-4811 (ext. 2087); Fax: 81-561-63-3532.

(^1)
The abbreviations used are: GlcNSO(3), N-sulfoglucosamine; PAPS, adenosine 3`-phosphate 5`-phosphosulfate; GlcA, glucuronic acid; IdoA, iduronic acid; HexA, hexuronic acid; CDSNS-heparin, completely desulfated and N-resulfated heparin; DeltaDi-OS, 2-acetamide-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid)-D-glucose; DeltaDi-6S, 2-acetamide-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; DeltaDi-NS, 2-deoxy-2-sulfamino-4-O-(4-deoxyalpha-L-threo-hex-4-enepyranosyluronic acid)-D-glucose; DeltaDi-(N,6)diS, 2deoxy-2-sulfamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; DeltaDi-(N,U)diS, 2-deoxy-2-sulfamino-4-O-(4deoxy-2-O-sulfo-alpha-L-threo-hex-4-enepyranosyluronic acid)-D-glucose; DeltaDi-(N,6,U)triS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enepyranosyluronic acid)-6-O-sulfo-D-glucose; AMan, 2,5-anhydro-D-mannose (when a subscript R follows this abbreviation, this refers to the corresponding alditol formed by reduction of the compound with NaBH(4)); DTT, dithiothreitol; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Mes, 4-morpholinoethanesulfonic acid.

(^2)
H. Habuchi, O. Habuchi, and K. Kimata, unpublished observations.


ACKNOWLEDGEMENTS

-We thank Dr. Sakaru Suzuki for helpful suggestions throughout this study, Yoko Noda for the excellent technical assistance, and Dr. Masaki Yanagishita for useful suggestions.


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