Recognition of Sulfation Pattern of Chondroitin Sulfate by Uronosyl 2-O-Sulfotransferase*

We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [35S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [35S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be ΔHexA-GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO4)-GlcA-GalNAc(6SO4)-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO4)-GlcA-GalNAc(6SO4) and GlcA-GalNAc(4SO4)-GlcA-GalNAc(6SO4) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.

We have shown previously that a highly sulfated trisaccharide structure containing both GalNAc(4,6-SO 4 ) and GlcA(2SO 4 ) residues, Gal-NAc(4,6-SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 ), exists at the nonreducing terminal of CS-A and that this structure was synthesized from a unique sequence, GalNAc(4SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 ), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST, gene name GALNAC4S-6ST) (22). Biosynthesis and functional roles of the highly sulfated nonreducing terminal structure has remained to be studied. We found previously that 6-O-sulfation of the GalNAc(4SO 4 ) residue at the nonreducing end by GalNAc4S-6ST was markedly stimulated by 2-O-sulfation of the penultimate GlcA residue (22), suggesting that 2-O-sulfation of the GlcA residue may occur prior to 6-O-sulfation of the nonreducing terminal GalNAc(4SO 4 ). Uronosyl 2-O-sulfotrasferase (2OST, gene name UST) (23), which was shown previously to catalyze 2-O-sulfation of GlcA and IdoA residues contained in CS and DS, respectively, may be involved in synthesis of these sequences. However, the mechanism by which these sequences are generated has not been fully understood because the strict substrate specificity of 2OST, especially recognition of the sulfation pattern around the targeting GlcA residue, has remained largely unclear. In the present study, we determined acceptor substrate specificity of the affinity purified recombinant 2OST using various glycosaminoglycans and oligosaccharides derived from chondroitin sulfate, and found that 2OST preferentially sulfated the GlcA residue located in the sequence GalNAc(4SO 4 )-GlcA-GalNAc(6SO 4 ). * This work was supported by Grants 14082206 and 16-4208 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a special research fund from the Seikagaku Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Tel.: 81-566-26-2642; Fax: 81-566- 26-2649; E-mail: ohabuchi@auecc.aichi-edu.ac.jp.
Preparation of the Recombinant Human 2OST-A DNA fragment that codes for the full open reading frame of 2OST (23) was amplified by PCR. The first PCR was carried out using oligonucleotides 2OST-F1, GGGTGACCTTTTCCTGGCAC, and 2OST-R1, GTCCCTTAAG-GATTTTACTTCCCCAAAC, which were synthesized according to the sequence of the 2OST cDNA clone (GenBank accession number AB020316), as primers, and the QUICK-Clone cDNA derived from human pancreas (Clontech) as a template. Amplification was carried out by 30 cycles of 94°C for 45 s, 55°C for 1 min, and 72°C for 3 min.
The second PCR was carried out using oligonucleotides 2OST-F2, CAGGAATTCGATGAAGAAGAAGCAGCAGCATC, and 2OST-R2, CGCAGATCTTCACCTCTTATAAATATCTTCCAGCCA, as primers, and the first PCR mixture as the template. At the 5Ј-ends of oligonucleotides 2OST-F2 and 2OST-R2, restriction enzyme recognition sites were introduced; an EcoRI site for 2OST-F2 and a BglII site for 2OST-R2. Amplification was carried out by 30 cycles of 94°C for 45 s, 52°C for 1 min, and 72°C for 2.5 min. The reaction products were subjected to agarose gel electrophoresis. The amplified DNA band was cut out and the DNA fragment was recovered from the gel, digested with EcoRI and BglII, and subcloned into these sites of pFLAG-CMV-2 plasmid (Eastman Kodak). Recombinant 2OST was expressed in COS-7 cells as a fusion protein with FLAG peptide and affinity purified (29). The purified protein was visualized with Western blot as described below before or after N-glycosidase F digestion (Fig. 1). After N-glycosidase F digestion, a single protein band was detected at the migration position of 49 kDa that agreed well with the molecular mass, 48,909, calculated from the cDNA.
Western Blot Analysis-The affinity purified 2OST was precipitated with 2 volumes of ethanol containing 1.3% (w/v) potassium acetate and digested with recombinant N-glycosidase F (Roche Molecular Biochemicals) by the methods recommended by the manufacturer. After digestion, the samples were separated by SDS-polyacrylamide gel electrophoresis as described by Laemmli (30). The separated proteins were

Disaccharide compositions of glycosaminoglycans that served as the acceptor for 2OST
Each glycosaminoglycan (25 nmol as galactosamine) was digested with chondroitinase ACII (CS-A, CS-C, and chondroitin) or ABC (DS and desulfated DS) and subjected to SAX-HPLC. Disaccharide compositions were determined by absorbance at 232 nm of unsaturated disaccharides. electrophoretically transferred to a Hybond ECL membrane (Amersham Biosciences), and stained with anti-FLAG M2 monoclonal antibody (Sigma). The blot was developed with polyclonal anti-mouse IgG antibody coupled to horseradish peroxidase using an ECL detection kit and a Hyperfilm ECL (Amersham Biosciences). Assay of Sulfotransferase Activity-2OST activity was assayed by the method described previously (23). The standard reaction mixture contained, in a final volume of 50 l, 2.5 mol of imidazole-HCl, pH 6.8, 2.6 g of protamine chloride, 25 nmol (as galactosamine) of CS-A, 50 pmol of [ 35 S]PAPS (about 5.0 ϫ 10 5 cpm), and enzyme. The reaction mixtures were incubated at 37°C for 30 min and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. 35 S-Labeled glycosaminoglycans were isolated by precipitation with ethanol followed by gel chromatography with a Fast Desalting Column as described previously (31), and radioactivity was determined. For determining the activity toward various glycosaminoglycans, CS-A was replaced with 25 nmol (as galactosamine for chondroitin, CS-C, DS, and oligosaccharides, or glucosamine for heparan sulfate, completely desulfated N-resulfated heparin, and keratan sulfate) of glycosaminoglycans or oligosaccharides. When oligosaccharides were used as the acceptors, the reaction mixtures were applied directly to the Superdex 30 column as described below, and the 35 S-labeled oligosaccharides were separated from 35

SO 4 and [ 35 S]PAPS.
Digestion with Chondroitinase ACII, Chondroitinase ABC, Chondro-6-sulfatase, Chondro-4-sulfatase, and ␤-Glucuronidase-Unless otherwise stated, digestion with chondroitinase ACII or chondroitinase ABC under standard conditions was carried out for 4 h at 37°C in the reaction mixture containing, in a final volume of 25 l, 1.25 mol of Tris acetate buffer, pH 7.5, 2.5 g of bovine serum albumin, and 30 milliunits of chondroitinase ACII or chondroitinase ABC. For degrading 35 S-labeled trisaccharides and tetrasaccharides with chondroitinase ABC, a strong condition was used under which digestion with chondroitinase ABC was carried out in the reaction mixtures described above three times successively; first with 120 milliunits of enzyme for 28 h, second with 100 milliunits of enzyme for 18 h, and finally with 100 milliunits of enzyme for 7 h. The new enzymes were added after heating the reaction mixtures at 100°C for 1 min. Digestion with chondro-6-sulfatase of Tetra A or Tetra B was carried out for 5 h at 37°C in the reaction mixture containing, in a final volume of 25 l, 1.25 mol of Tris acetate buffer, pH 7.5, 2.5 g of bovine serum albumin, and 50 milliunits of chondro-6-sulfatase. After digestion of 35 S-labeled glycosaminoglycans or oligosaccharides with chondroitinase ACII or chondroitinase ABC, digestion with chondro-6-sulfatase or chondro-4-sulfatase was carried out twice successively in the reaction mixtures described above; first with 100 milliunits of enzyme for 17 h and second with 100 milliunits of enzyme for 5 h. Digestion with ␤-glucuronidase was carried out for 4 h at 37°C in a reaction mixture containing, in a final volume of 40 l, 35 S-labeled tetrasaccharide (ϳ40 nmol as galactosamine), 2 mol of sodium acetate buffer, pH 4.5, 20 nmol of 2-acetamido-2-deoxy-D-galactonic acid-1,4-lactone, 0.8 mol of sodium fluoride, and 18 units of ␤-glucuronidase. Under these conditions, removal of the nonreducing terminal GlcA was complete and no release of inorganic sulfate was observed.
Removal of Unsaturated Uronic Acid by Mercuric Acetate Treatment-Removal of unsaturated uronic acid was carried out as described (32). 35 S-Labeled or -unlabeled tetrasaccharide containing unsaturated uronic acid were dried and dissolved in 0.5 ml of 35 mM mercuric acetate in 25 mM Tris with 25 mM sodium acetate, pH 5.0. The reaction was carried out for 1 h at room temperature. After the reaction was over, the samples were applied to a Dowex 50 (H ϩ ) column (bed volume of 1 ml).
The column was washed with 3 ml of water. The flow-through fractions and the washings were combined and lyophilized. The lyophilized materials were further purified with Superdex 30 and SAX-HPLC.
Sulfation of the Trisaccharide, Which Was Derived from Tetra A by Mercuric Acetate Treatment, with GalNAc4S-6ST-The sulfotransferase reaction was carried out as described (26,33). The reaction mixture contained, in a final volume of 50 l, 2.5 mol of imidazole-HCl, pH 6.8, 1 mol of CaCl 2 , 1 mol of reduced glutathione, the 35 S-labeled trisaccharide derived from Tetra A by mercuric acetate treatment (about 6000 cpm), 1.35 nmol of Oligo I (final 27 M), 100 nmol of unlabeled PAPS (final 2 mM), and 180 ng of the purified squid GalNAc4S-6ST. The reaction mixture was incubated at 15°C for 24 h and the reaction was stopped by immersing the reaction tube in a boiling water bath for 1 min. After the reaction was stopped, the reaction mixture was applied to the Superdex 30 column. Fractions containing the radioactive oligosaccharide were collected, lyophilized, and separated with SAX-HPLC.
Superdex 30 Chromatography and HPLC-A Superdex 30 16/60 column was equilibrated with 0.2 M NH 4 HCO 3 , and run at a flow rate of 2 ml/min. One-ml fractions were collected. Separation of the degradation products formed from 35 S-labeled glycosaminoglycans and 35 S-labeled oligosaccharides were carried out by HPLC using a Whatman Partisil-10 SAX column (4.6 ϫ 25 cm) equilibrated with 5 mM KH 2 PO 4 . The column was developed with a gradient (5 mM KH 2 PO 4 for 10 min followed by a linear gradient from 5 to 500 mM KH 2 PO 4 ). Fractions (0.5 ml) were collected at a flow rate of 1 ml/min and a column temperature of 40°C.

Sulfation of Various Glycosaminoglycans by the Recombinant 2OST-
The rates of transfer of sulfate to various glycosaminoglycans were determined (TABLE TWO). Among these glycosaminoglycans, desulfated DS was the best acceptor. CS-A from whale cartilage and sturgeon notochord were both better acceptors than CS-C. The rate of sulfation of chondroitin was much lower than the rate of sulfation of CS-A. Unlike a previous report (23), DS was a poor acceptor. The rate of sulfation of DS was increased when the concentration of protamine was increased as described previously in the activity of C4ST-1 (34). CS-E, keratan sulfate, heparan sulfate, and completely desulfated re-N-sulfated heparin hardly served as acceptor. To determine the position to which sulfate was transferred, the 35 S-labeled glycosaminoglycans  NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 derived from whale cartilage CS-A and CS-C were digested with chondroitinase ACII and subjected to SAX-HPLC (Fig. 2, A and C). The radioactivity was exclusively detected at the position of ⌬Di-diS D . After chondro-6-sulfatase digestion, the radioactivity was shifted to the position of ⌬Di-2S (Fig. 2, B and D). The same results were obtained for sturgeon notochord CS-A (data not shown), in which the GalNAc(6SO 4 ) residue was less than 5% of the total GalNAc residues (TABLE ONE). The observation that 2OST transferred sulfate exclusively to position 2 of GlcA residues adjacent to the nonreducing side of GalNAc(6SO 4 ) residue, even when sturgeon notochord CS-A was used as the acceptor, strongly suggests that 2OST should require the presence of the GalNAc(6SO 4 ) residue at the reducing side of the targeted GlcA. When 35 S-labeled glycosaminoglycans derived from chondroitin were digested with chondroitinase ACII, more than 90% of the radioactivity was recovered in ⌬Di-2S (data not shown), indicating that 2OST could transfer sulfate to the GlcA residue adjacent to the nonreducing side of the GalNAc residue, albeit at a much lower rate. When 35 Slabeled glycosaminoglycans derived from desulfated DS were digested with chondroitinase ABC, the radioactivity was detected at the position of ⌬Di-2S (Fig. 3A). We observed previously that, when desulfated DS was used as the acceptor for C4ST-1, sulfate was mainly transferred to position 4 of the GalNAc residue located in the GlcA-rich region (34,35). To see if 2OST also prefers GlcA residues contained in desulfated DS, the [ 35 S]glycosaminoglycans formed from desulfated DS by the reaction with 2OST was digested with chondroitinase ACII; however, no disaccharide products were obtained (data not shown). These results indicate that 2OST has no preference to GlcA residue in the desulfated DS. When 35 S-labeled glycosaminoglycans derived from DS in the presence of 26.3 g of protamine were digested with chondroitinase ABC, the major radioactivity was detected at the position of ⌬Di-diS B (Fig.  3B). After chondro-4-sulfatase digestion, ⌬Di-diS B disappeared and shifted to ⌬Di-2S (Fig. 3C). These observations indicate that, unlike the GlcA residue, IdoA residues adjacent to GalNAc residues could be sulfated efficiently by 2OST. In contrast, the rate of sulfation of the IdoA residue adjacent to the GalNAc(4SO 4 ) residue was almost below the detectable level under standard assay conditions.

Characterization of a Tetrasaccharide Obtained from 35 S-Labeled Glycosaminoglycans Derived from CS-A by a Limited Digestion with
Chondroitinase ACII-We have shown previously that GlcA(2SO 4 )containing sequences in CS exhibited various degrees of resistance to chondroitinase ACII (22). To clarify the sequence around the 2-O-sulfated GlcA residue, we tried to prepare oligosaccharides derived from the 2-O-sulfated regions by limited digestion with chondroitinase ACII. When 35 S-labeled glycosaminoglycans derived from CS-A was subjected to a limited digestion with chondroitinase ACII and separated with Superdex 30 chromatography, a radioactive peak appeared slightly before the position of the disulfated tetrasaccharide (Fig. 4A). As the conditions of the chondroitinase ACII digestion became stronger, the tetrasaccharide was converted to ⌬Di-diS D (Fig. 4, B-D). These observations indicate that 2OST transferred sulfate to a unique tetrasaccharide sequence in CS-A. When the tetrasaccharide fraction was separated with SAX-HPLC, a single radioactive peak was detected behind ⌬Di-diS E (Fig. 5A). This material was named Tetra A. After digestion with chondroitinase ACII under the standard conditions, radioactivity  of Tetra A was shifted to ⌬Di-diS D (Fig. 5B). After further digestion with chondro-6-sulfatase, the radioactive peak was shifted to ⌬Di-2S (Fig.  5C). When Tetra A was digested with chondro-6-sulfatase alone, a radioactive peak was detected slightly behind ⌬Di-diS D (Fig. 5D). This radioactive peak was shifted to ⌬Di-2S after further digestion with chondroitinase ACII (Fig. 5E). Because chondro-6-sulfatase could remove sulfate from the GalNAc(6SO 4 ) residue located exclusively to the reducing end of the oligosaccharides (22,36), these observations indicate that Tetra A should contain the GlcA(2SO 4 )-GalNAc(6SO 4 ) unit at the reducing side. When Tetra A was treated with mercuric acetate, the resulting trisaccharide behaved identically with Oligo I in SAX-HPLC (Fig. 6B). When the trisaccharide was incubated with squid GalNAc4S-6ST in the presence of 2 mM nonradioactive PAPS, the radioactivity was quantitatively shifted to the position of Oligo II (Fig.  6C). We found previously that squid GalNAc4S-6ST could transfer sulfate to position 6 of both nonreducing terminal and reducing terminal GalNAc(4SO 4 ) residues of the 4-sulfated trisaccharide (26). Because the reducing terminal residue of Tetra A was found to be GalNAc(6SO 4 ) as described above, conversion of the trisaccharide to a material corresponding to Oligo II by the reaction with squid GalNAc6S-6ST indicates that the nonreducing terminal residue of the trisaccharide should be GalNAc(4SO 4 ). The structural analysis of Tetra A is summarized in Scheme 1. Taken together, it is most probable that the trisaccharide is identical to Oligo I and thus Tetra A is ⌬HexA-GalNAc(4SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 ).
Isolation and Characterization of Tetrasaccharides Containing GlcA(2SO 4 ) from CS-A-To confirm the deduced structure of Tetra A, we tried to isolate a tetrasaccharide from CS-A that behaved identically with Tetra A in SAX-HPLC. CS-A (550 mol as galactosamine) was digested for 4 h at 37°C with chondroitinase ACII in the reaction mixture containing, in a final volume of 10 ml, 500 mol of Tris acetate buffer, pH 7.5, 1 mg of bovine serum albumin, and 2 units of chondroitinase ACII. The degradation products were separated with Superdex 30 chromatography (Fig. 7A). Tetrasaccharide fractions were collected and further separated with SAX-HPLC (Fig. 7B). A peak was detected at the position of Tetra A. This tetrasaccharide was further purified by Superdex 30 chromatography. About 0.4 mol (as galactosamine) of the tetrasaccharide was obtained from 550 mol of whale cartilage CS-A. When the tetrasaccharide was digested with chondroitinase ACII, ⌬Di-diS D and ⌬Di-4S were obtained (Fig. 8B). After further digestion with chondro-6-sulfatase, ⌬Di-diS D was shifted to ⌬Di-2S (Fig. 8C), whereas with further digestion with chondro-4-sulfatase, ⌬Di-4S was shifted to ⌬Di-0S (data not shown). When the tetrasaccharide was digested with chondro-6-sulfatase alone, a peak was obtained slightly behind ⌬Di-diS D (Fig. 8D); the retention time of this peak was strictly the same as the retention time of the radioactive product formed from Tetra A after chondro-6-sulfatase digestion (Fig. 5D). After further digestion with chondroitinase ACII, this peak was converted to ⌬Di-4S and ⌬Di-2S   (Fig. 8E). These observations clearly indicate that the tetrasaccharide is ⌬HexA-GalNAc(4SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 ). The sequence of the tetrasaccharide was confirmed by another approach. When the tetrasaccharide was subjected to mercuric acetate treatment, a trisaccharide was obtained whose retention time was strictly the same as that of Oligo I. When the trisaccharide was digested with chondroitinase ABC and separated with SAX-HPLC, GalNAc(4SO 4 ) and ⌬Di-diS D were formed (data not shown). The structural analysis of the tetrasaccharide is summarized in Scheme 2. From the analytical data of Tetra A and the tetrasaccharide that behaved identically with Tetra A, we concluded that Tetra A is identical to the tetrasaccharide, and thus 2OST transferred sulfate to position 2 of the GlcA residue located in the unique sequence, GalNAc(4SO 4 )-GlcA-GalNAc(6SO 4 ), present in CS-A.
Sulfation of Various CS-derived Oligosaccharides by the Recombinant 2OST-As described above, 2OST preferentially transferred sulfate to position 2 of the GlcA residue in the unique sequence GalNAc(4SO 4 )-

FIGURE 7. Preparation and separation of a tetrasaccharide from CS-A that behaved identically with Tetra A.
A, CS-A was digested with chondroitinase ACII as described in the text. The digest was applied to the Superdex 30 column. The column was monitored at 232 nm. The fractions indicated by a horizontal bar were collected, concentrated, and lyophilized. The standards were the same as those described in the legend to Fig. 4. B, the lyophilized materials were separated with SAX-HPLC as described under "Experimental Procedures." The column was monitored at 232 nm. The broken line depicts the concentration of KH 2 PO 4 . The standards were the same as those described in the legend to Fig.  2. The elution position of Tetra A was also indicated. The material eluted at the position of Tetra A was used for the following experiments.  GlcA-GalNAc(6SO 4 ). Thus, 2OST appears to recognize GalNAc(4SO 4 ) and GalNAc(6SO 4 ) residues neighboring the nonreducing side and reducing side, respectively, of the acceptor GlcA residue. This substrate specificity of 2OST was further confirmed by sulfation of various trisaccharides and tetrasaccharides with different sulfation patterns. As shown in TABLE THREE, Tetra-46 (GlcA-GalNAc(4SO 4 )-GlcA-GalNAc(6SO 4 )) and Tri-46 (GalNAc(4SO 4 )-GlcA-GalNAc(6SO 4 )) were the best acceptors among tetrasaccharides and trisaccharides, respectively. Tetra-46 was a better acceptor than Tri-46. No sulfate incorporation was detected under the same conditions when nonsulfated trisaccharide and tetrasaccharide were used as acceptors (data not shown). When 2-O-sulfated Tetra-46 was separated with SAX-HPLC, a single radioactive peak was detected at the retention time earlier than the retention time of Tetra A (Fig. 9A). This radioactive peak was shifted to the position of Oligo I after ␤-glucuronidase digestion (Fig. 9B), indicating that sulfate was transferred to the GlcA residue between GalNAc(4SO 4 ) and GalNAc(6SO 4 ) but not to the nonreducing terminal one. When 2-O-sulfated Tetra-46 was digested with chondroitinase ABC and subjected to SAX-HPLC, radioactivity was exclusively detected at the position of ⌬Di-diS D (Fig. 9C), and shifted to the position to ⌬Di-2S after chondro-6-sulfatase digestion (Fig. 9D). These observations are well consistent with the substrate specificity of 2OST revealed by the analysis of the 2-O-sulfated CS-A. The K m and V max for Tetra-46, Tetra-44 (GlcA-GalNAc(4SO 4 )-GlcA-GalNAc(4SO 4 )), and Tetra-66 (GlcA-GalNAc(6SO 4 )-GlcA-GalNAc(6SO 4 )) were compared (TABLE  FOUR). It is evident that the sulfation pattern of the tetrasaccharides markedly affected their affinities to 2OST; Tetra-46 showed the highest affinity. These observations indicate that the selective sulfation of GlcA residues between GalNAc(4SO 4 ) and GalNAc(6SO 4 ) by 2OST seems to be due mainly to the high affinity of 2OST toward this sequence.

DISCUSSION
In this paper, we found that 2OST preferentially transferred sulfate to the GlcA residue located in the sequence, GalNAc(4SO 4 )-GlcA-GalNAc(6SO 4 ), in CS-A. CS-D from shark cartilage was reported to   contain the GlcA(2SO 4 )-GalNAc(6SO 4 ) unit at the reducing side of the GlcA-GalNAc(4SO 4 ) unit (37)(38)(39). CS-D in the brain and cartilage disappeared in C6ST Ϫ/Ϫ mice (40). A spondyloepiphyseal dysplasia, Omani type, was identified to be associated with a mutation in the C6ST-1 gene. In the cells and urine of a patient, the HexA(2S)-GalNAc(6S) unit was significantly reduced (41). These observations are consistent with the specificity of 2OST described here. Chondroitin was much less acceptor than CS-A, indicating that pre-existing sulfate groups on the GalNAc residue stimulated 2OST activity. In contrast, desulfated DS was a much better acceptor than DS, suggesting that 2-O-sulfation of the IdoA residue may occur before 4-O-sulfation of the GalNAc residue. 2OST was cloned on the basis of the sequence homology with heparan sulfate 2-sulfotransferase (HS2ST, gene name HS2ST) (23,42). 2-O-Sulfation of uronate in heparan sulfate is assumed to be catalyzed by a single HS2ST, because only one HS2ST gene has been detected in any of the vertebrate examined to date (43), and 2-O-sulfated uronates in heparan sulfate were not detected in HS2ST-deficient mice (44). Isoforms of 2OST also have not been found to date, suggesting that 2OST may be involved in biosynthesis of both CS and DS, although recognition of the sulfation pattern by 2OST is different between these glycosaminoglycans.
The sulfation profiles of various glycosaminoglycans by the affinity purified 2OST observed here were similar to the results previously reported (23); however, some significant differences are present. Unlike the previous report, the affinity purified recombinant 2OST sulfated CS-A, CS-C, and desulfated DS much more efficiently than DS. The poor acceptor activity of DS is not because of the source of DS, because a similar low activity was observed when DS from porcine intestinal mucosa was used as the acceptor (data not shown). It is not clear why the apparent specificity of the affinity purified 2OST expressed in COS-7 cells is so different from that of the crude 2OST expressed in Sf9 insect cells. Post-translational modification of the expressed 2OST might affect its substrate specificity. Alternatively, the high activity of the crude 2OST toward DS might be because of the host cell-derived stimulating factors, because we observed that high concentrations of protamine stimulated 2OST activity toward DS.
The recognition of sulfation pattern by 2OST was also demonstrated by comparison of the rate of sulfation of oligosaccharides with defined sulfation pattern. Tri-46 and Tetra-46 served as efficient acceptors, whereas Tri-64 and Tetra-64 did not. Tri-66, Tri-44, Tetra-66, and Tetra-44 were sulfated much more slowly than Tri-46 and Tetra-46. These observations are well consistent with the specificity of 2OST revealed by the sulfation of CS-A. Because the rate of sulfation of Tetra-46 was as high as that of glycosaminoglycans, the polysaccharide nature of the acceptors may not necessarily be required by 2OST as observed in GalNAc4S-6ST (29).
We have shown previously that a trisaccharide sequence, GalNAc(4SO 4 )-GlcA(2SO 4 )-GalNAc(6SO 4 )-, is present at the nonreducing terminal of CS-A (22). 2OST may be involved in the synthesis of the nonreducing terminal sequence because Tri-46 was a good acceptor for 2OST. However, when CS-A was used as the acceptor for 2OST, sulfate was transferred to the internal region of CS-A, because Tetra A, which bears unsaturated uronate at the nonreducing end and hence should be released from the internal region of the sulfated CS-A, was obtained as a sole intermediate product from the sulfated CS-A by the limited digestion with chondroitinase ACII. Because the rate of 2-Osulfation of Tri-46 was lower than the rate of 2-O-sulfation of Tetra-46, 2-O-sulfation of the nonreducing terminal may proceed much more slowly than 2-O-sulfation of the internal sequence. Alternatively, the sequence of GalNAc(4SO 4 )-GlcA-GalNAc(6SO 4 )-might be very rare at the nonreducing terminal of CS-A.
In the present paper, we successfully prepared radioactive Oligo I through the sulfation of Tetra-46 with the recombinant 2OST followed by ␤-glucuronidase digestion. On the other hand, we described previously that radioactive Oligo II could be prepared from Oligo I by reaction with GalNAc4S-6ST (22). These radioactive oligosaccharides may be useful probes for elucidating the functional roles of these highly sulfated structures.