Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene.

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO(4))) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO(4)) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO(4)) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from PAPS to position 6 of GalNAc(4SO 4 ) residue and appears to be a key enzyme for the synthesis of GalNAc(4,6-bisSO 4 ) residues. This enzyme activity was found in hen oviduct (25), squid cartilage (26), quail oviduct (27), and human serum (28). Recently, the squid cartilage GalNAc4S-6ST was purified to apparent homogeneity (29). The purified GalNAc4S-6ST was found to transfer sulfate to mainly position 6 of internal GalNAc(4SO 4 ) residue when CS-A or DS were used as acceptors. However, it has not been clear whether sulfotransferases with the substrate specificity similar to the specificity of squid GalNAc4S-6ST are present in the vertebral tissues. In this paper, we report identification of the human GalNAc4S-6ST cDNA and characterization of the recombinant enzyme expressed from the cDNA. Surprisingly, we found that the nucleotide sequence of the human GalNAc4S-6ST cDNA is nearly identical to the sequence of B cell RAG-associated gene (30), suggesting an interesting possibility that human GalNAc4S-6ST might be involved in the regulation of recombination activating gene, and thereby in the maturation of B cells.
* This work was supported by Grants-in-aid for Scientific Research on Priority Areas 05274107 and 10178102 from the Ministry of Education, Science, Sports and Culture of Japan; by grants-in-aid from the Mizutani Foundation for Glycoscience; and by a special research fund from Seikagaku Corp. 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.

Materials-
[ 35 S]PAPS was prepared as described (31). Chondroitin (squid skin) was prepared as described (32). Keratan sulfate (bovine cornea) was a generous gift from Seikagaku Corporation. CS-E (squid cartilage), which was eluted with 1.5 M NaCl from DEAE-Sephadex A-50, was prepared as described (33). A trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were prepared from CS-A by digestion with testicular hyaluronidase and ␤-glucuronidase (29). Analytical data of glycosaminoglycans and the 4-sulfated oligosaccharides have been shown in a previous paper (29). Molar ratios of the nonreducing terminal GalNAc(4SO 4 ) residue to the internal GalNAc(4SO 4 ) residue were determined by SAX-HPLC after chondroitinase ACII or ABC digestion as described previously (29). CS-A and DS (625 nmol as galactosamine) were digested with chondroitinase ACII and chondroitinase ABC, respectively. The digested materials (25 nmol as galactosamine for determination of ⌬Di-4S and 500 nmol for determination of GalNAc(4SO 4 )) were subjected to SAX-HPLC together with Na 2 35 SO 4 as an internal standard. GalNAc(4SO 4 ) and ⌬Di-4S were monitored by absorption at 210 nm ( Fig. 5). At 210 nm, the observed ratio of (molecular absorption of monosaccharides)/(molecular absorption of unsaturated disaccharides) was 0.32 (29). From the elution profiles, molar ratios of GalNAc(4SO 4 ) to ⌬Di-4S were calculated. A nonsulfated trisaccharide and a nonsulfated pentasaccharide were prepared from desulfated CS-A by digestion with testicular hyaluronidase and ␤-glucuronidase (34).
Determination of Amino Acid Sequence of the Purified Protein-The squid GalNAc4S-6ST was purified from the squid cartilage as described previously (29). A portion of the purified GalNAc4S-6ST (10 g as protein) was subjected to SDS-polyacrylamide gel electrophoresis (10% gel) according to the method of Laemmli (35) after reduction and denaturation in loading buffer containing 5% (v/v) 2-mercaptoethanol. The polyacrylamide gel was stained with Coomassie Brilliant Blue, and the band of the 63-kDa protein was excised. The excised gel containing the protein was sent to Apro Science Co. Ltd. (Naruto, Japan) for the amino-terminal amino acid sequencing of peptides separated by reverse phase HPLC after limited digestion of the protein with proteinase Lys-C.
Preparation of Poly(A) ϩ RNA from the Squid Cartilage-Squid cranial cartilage was dissected, freed of soft tissues by wiping with cotton cloth, and put into liquid nitrogen. The frozen cartilage was ground to powder in a mortar in the presence of liquid nitrogen. The cartilage powder was placed in 10 volumes of an ice-cold guanidine thiocyanate solution and homogenized with a Polytron homogenizer. The homogenate was centrifuged at 100,000 ϫ g for 30 min. The clear supernatant fraction was used for isolation of total RNA by the guanidine thiocyanate/CsCl methods (36). Poly(A) ϩ RNA was purified by oligo(dT)-cellulose column chromatography.
Oligonucleotides and Polymerase Chain Reaction-Degenerate oligonucleotide primers were designed as indicated in Fig. 1A. Two sense primers (5s-1 and 5s-2) and two antisense primers (4a-1 and 4a-2) were prepared from the amino acid sequences of peptide 5 and peptide 4, respectively. The first strand of cDNA was synthesized by the reverse transcriptase reaction using poly(A) ϩ RNA from the squid cartilage as a template and oligo(dT) or random oligonucleotide as a primer. The reverse transcriptase reaction mixture contained, in a final volume of 20 l, 2 g of poly(A) ϩ RNA, 0.5 g of oligo(dT) or random primers, 10 mM dithiothreitol, 0.5 mM each of four deoxynucleoside triphosphates, 30 units of RNase inhibitor (Takara), and 400 units of reverse transcriptase (Superscript II, Life Technologies, Inc.). The reaction was carried out at 42°C for 50 min. After the reaction was stopped by heating at 70°C for 15 min, 0.5 unit of RNase H (Life Technologies, Inc.) was added and incubated for 20 min at 37°C. The PCR reaction was carried out in a final volume of 25 l containing 25 pmol each of the oligonucleotide primers (5s-1 and 4a-2), 1 l of the reverse transcriptase reaction mixture in which the first strand cDNA was synthesized, 0.2 mM each of four deoxynucleoside triphosphates, and 1.5 units of Taq polymerase (Qiagen). Amplification was carried out by 40 cycles of 94°C for 45 s, x°C for 1.5 min, and 72°C for 1 min. The annealing temperature (x°C) was decreased from 48°C to 32°C by 4°C/3 cycles, and retained at 28°C for an additional 25 cycles. The second PCR was carried out using the reaction mixture of the first PCR as a template and oligonucleotide primer 5s-2 and 4a-1 under the same reaction conditions as the first PCR. Reaction products of the second PCR were subjected to agarose gel electrophoresis (Fig. 1B, lane 1). The amplified DNA band (indicated by an arrowhead in Fig. 1B) was cut out, and the DNA fragment was recovered from the gel, and cycle sequenced using primer 5s-2 and 4a-1 as sequence primers. The nucleotide sequence was determined by the dideoxy chain termination method using a DNA sequencer (Applied Biosystems model 373A).
Construction of pcDNAGalNAc4S-6ST-To construct the plasmid containing the human GalNAc4S-6ST cDNA named pcDNAGalNAc4S-6ST, the pBluescript II plasmid containing the human GalNAc4S-6ST cDNA (accession no. AB011170, gene no. KIAA 0598), which was a generous gift from Kazusa DNA Research Institute (Kisarazu, Japan), was cut with EcoRI and the fragment with 2258 nucleotides was ligated into EcoRI site of pcDNA3 (Invitrogen).
Transient Expression of the Human GalNAc4S-6ST cDNA in COS-7 Cells-COS-7 cells were transfected with pcDNAGalNAc4S-6ST using the DEAE-dextran method as described previously (37,38). After transfection, the cells were extracted with 0.15 M NaCl, 10 mM Tris-HCl, pH 7.2, 10 mM MgCl 2 , 2 mM CaCl 2 , 0.5% Triton X-100, and 20% glycerol for 30 min on a rotatory shaker. The extracts were centrifuged at 10,000 ϫ g for 10 min. The sulfotransferase activities in the supernatant fractions were measured using CS-A or the 4-sulfated trisaccharide as acceptors.
Construction of pFLAGGalNAc4S-6ST and Preparation of the Affinity-purified Protein-Recombinant GalNAc4S-6ST was expressed as a fusion protein with FLAG peptide and was affinity-purified. A DNA fragment that codes for full open reading frame was amplified by PCR using the human GalNAc4S-6ST cDNA (accession no. AB011170) as a template. The 5Ј and 3Ј primers were CGCAAGCTTATGAGGCACTG-CATTAATTGCTGC and CAGGAATTCTCACGTCGTCTTCCACGCAA-AC, respectively. At the 5Ј end of the oligonucleotide primers, restriction enzyme recognition sites were introduced: HindIII site for the sense primer and EcoRI site for the antisense primer. The PCR product was digested with EcoRI and HindIII and subcloned into these sites of pFLAG-CMV-2 plasmid (Eastman Kodak Co.). The resulting plasmid was transfected in COS-7 cells, and the fusion protein produced was extracted as described above. The cellular extracts from 10 10-cm dishes were applied to an anti-FLAG monoclonal antibody-conjugated agarose column (0.5 ml) (Sigma). The absorbed materials were eluted with 1.5 ml of a buffer containing FLAG peptide under the conditions recommended by the manufacturer.
Assay of Sulfotransferase Activity-GalNAc4S-6ST activity was assayed by the method described previously (29) with a slight modification. The standard reaction mixture contained, in a final volume of 50 l, 2.5 mol of imidazole-HCl, pH 6.8, 0.5 mol of CaCl 2 , 1 mol of reduced glutathione, 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 20 min, and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. After the reaction was stopped, 35 S-labeled glycosaminoglycans were isolated by the precipitation with ethanol followed by gel chromatography with a fast desalting column as described previously (39) and radioactivity was determined. For determining the activity toward various glycosaminoglycans, CS-A was replaced with 25 nmol (as galactosamine for CS-C, DS, 4-sulfated oligosaccharides, and nonsulfated oligosaccharides; or glucosamine for heparan sulfate, CDSNS-heparin, and keratan sulfate) of glycosaminoglycans or oligosaccharides. For determining the position of sulfate transferred to CS-A and DS, 35 S-labeled glycosaminoglycans were digested with chondroitinase ACII, chondroitinase ABC, chondroitinase ACII plus chondro-6-sulfatase, or chondroitinase ABC plus chondro-6-sulfatase as described previously (29). The radioactive products formed after the enzymatic digestion were separated with HPLC using a Whatman Partisil-10 SAX column as described below, and 35 S radioactivity was determined. When oligosaccharides were used as 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 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 mm ϫ 25 cm) equilibrated with 10 mM KH 2 PO 4 . The column was developed with 10 mM KH 2 PO 4 for 10 min, followed by a linear gradient from 10 to 450 mM KH 2 PO 4 as indicated in Fig. 4. Fractions (0.5 ml) were collected at a flow rate of 1 ml/min and a column temperature of 40°C.

Identification of a Human cDNA Showing Sequence
Homology to the Squid GalNAc4S-6ST-We obtained amino acid sequences of five peptides from the purified squid GalNAc4S-6ST (Table I). When primer 5s-1 and 4a-2 (for the nucleotide sequences of the primers, see Fig. 1A) designed from the amino acid sequences of peptides 5 and 4, respectively, were used in a PCR with poly(A) ϩ RNA from squid cartilage as a template, no amplification of DNA was observed (data not shown); however, a DNA fragment of about 380 base pairs was clearly amplified on the second PCR, in which the reaction mixture of the first PCR was used as a template and oligonucleotide 5s-2 and 4a-1 were used as primers (Fig. 1B, lane 1). When the amino acid sequence deduced from the nucleotide sequence of the amplified DNA was used for FASTA search, we found a human cDNA clone (accession no. AB011170) located to chromosome 10 that showed significant homology to the squid DNA. Comparison of the sequence of the human cDNA clone with the amplified squid DNA revealed ϳ40% identity and ϳ61% similarity at the amino acid level (Fig. 2). This clone contained an open reading frame that predicts a protein composed of 561 amino acids. Examining the amino acid sequence of this protein revealed the presence of a transmembrane domain of type II topology and putative PAPS binding domains (5Ј-PSB and 3Ј-PB) (Fig. 2), suggesting that this cDNA may encode a novel sulfotransferase. In addition, the four of five peptides derived from the purified squid GalNAc4S-6ST nearly matched the human protein (Fig. 2). Taken together, the results indicated that the protein deduced from the human cDNA may be a human counterpart for the purified squid GalNAc4S-6ST. We also found another human clone (accession no. AF026477) whose nucleotide sequence is nearly identical to the human cDNA with accession no. AB011170. The cDNA with accession no. AF026477 was reported as human B cell RAG-associated gene (hBRAG) (30).
Expression of the Human cDNA Showing Homology to the Squid GalNAc4S-6ST in COS-7 Cells-The human cDNA isolated from human brain (accession no. AB11170, gene no. KIAA 0598) (40), which had been found in the GenBank data base by FASTA search, was a generously gift from the Kazusa DNA Research Institute (Kisarazu, Japan). We confirmed that the nucleotide sequence of the open reading frame of this cDNA was strictly the same as the sequence that appeared in the data base. Evidence that the human cDNA encodes a novel sulfotransferase was obtained by expressing it in COS-7 cells. COS-7 cells were transfected with the pcDNAGalNAc4S-6ST, a recombinant plasmid containing the cDNA in a mammalian expression vector, pcDNA3 (Invitrogen). The transfected cells were scraped at 67 h after transfection, extracted with gentle shaking in a buffer containing 0.5% Triton X-100, and centrifuged. The sulfotransferase activities in the supernatant fractions were determined using CS-A or a trisaccharide having sulfate groups at position 4 of GalNAc residues as acceptors. As shown in Fig. 3, when the plasmid containing the human cDNA was used, the sulfotransferase activity toward the 4-sulfated trisaccharide was increased more than 5-fold over the control, although the sulfotransferase activity toward CS-A was increased only 2-fold. We hypothesized that this sulfotransferase might transfer sulfate predominantly to the nonreducing terminal sugar residue, because the 4-sulfated trisaccharide was a better acceptor than CS-A.
Substrate Specificity of the Recombinant Human Sulfotransferase-To determine the substrate specificity of the expressed enzyme, we prepared the affinity-purified protein from the extracts of COS-7 cells transfected with pFLAGGalNAc4S-6ST and the sulfotransferase activity was determined using various acceptors. In Table II, the sulfotransferase activity toward various glycosaminoglycans and oligosaccharides is shown. Among glycosaminoglycans, CS-A and DS were able to serve as acceptors. Chondroitin was a poor acceptor. CS-C, CS-E, keratan sulfate, heparan sulfate, and CDSNS-heparin hardly served as acceptors. Sulfation of oligosaccharides having GalNAc(4SO 4 ) residue at the nonreducing end (4-sulfated trisaccharide and 4-sulfated pentasaccharide) proceeded at much higher rates than the sulfation of CS-A. In contrast, the nonsulfated trisaccharide and nonsulfated pentasaccharide did not serve as acceptors at all.   35 S-Labeled Oligosaccharides-To determine the position of the sulfate group transferred to CS-A, we digested 35 S-labeled CS-A with chondroitinase ACII, and analyzed the digestion products by Partisil-10 SAX-HPLC (Fig. 4). When the 35 S-labeled CS-A was digested with chondroitinase ACII, the radioactivity was detected at the position of GalNAc(4,6-bisSO 4 ) and ⌬Di-diS E (Fig. 4A). To determine which sulfate group of GalNAc(4,6-bisSO 4 ) and ⌬Di-diS E had 35 S radioactivity, the degradation products obtained after chondroitinase ACII were further digested with chondro-6-sulfatase,  GalNAc4S-6ST COS-7 cells were transfected with pFLAGGalNAc4S-6ST or vector alone, and the affinity-purified protein was prepared as described under "Experimental Procedures." Sulfotransferase activities were assayed using various glycosaminoglycans and oligosaccharides as described under "Experimental Procedures." The activity of the affinity-purified fraction obtained from COS-7 cells transfected with the vector alone was less than 0.6 pmol/min/ml for any acceptor used and was subtracted from the individual activity.

Cloning of GalNAc 4-Sulfate 6-O-Sulfotransferase
and subjected to SAX-HPLC (Fig. 4B). After digestion with chondro-6-sulfatase, 35 S radioactivity was shifted to the position of inorganic sulfate, indicating that 35 SO 4 was transferred to position 6 of GalNAc(4SO 4 ) residues located at the nonreducing terminal and internal repeating units of CS-A at a nearly equal rate. On the other hand, 35 S-labeled DS was hardly depolymerized by chondroitinase ACII digestion (data not shown). When the 35 S-labeled DS was digested with chondroitinase ABC, a main radioactive peak was detected at the position of GalNAc(4,6-bisSO 4 ) and a small peak was detected at the position of ⌬Di-diS E (Fig. 4C). As observed in the 35 S-labeled CS-A, most of the radioactivity was shifted to the position of inorganic sulfate after chondro-6-sulfatase digestion (Fig. 4D). A small radioactivity remaining at the position of GalNAc(4,6-bisSO 4 ) after chondro-6-sulfatase digestion disappeared after further digestion with chondro-6-sulfatase. DS was thus sulfated mainly at position 6 of nonreducing terminal GalNAc(4SO 4 ) residue adjacent to iduronic acid residue. When chondroitin was used as an acceptor, the transfer of sulfate still occurred at the nonreducing terminal and internal GalNAc(4SO 4 ) residues (data not shown), although GalNAc(4SO 4 ) residues are only minor components of chondroitin. These results clearly indicate that the human cDNA showing homology to the squid DNA encodes human GalNAc4S-6ST. To clear the possibility that the lower acceptor efficiency of DS compared with CS-A may be because of the lower contents of nonreducing terminal GalNAc(4SO 4 ), we analyzed the molar ratios of the nonreducing terminal GalNAc(4SO 4 ) residue to the internal GalNAc(4SO 4 ) residue of these glycosaminoglycans by the SAX-HPLC as described under "Experimental Procedures." A peak corresponding to GalNAc(4SO 4 ) was clearly detected in the digests of both CS-A and DS (indicated by arrowheads in Fig. 5, B and C). The calculated molar ratio of the nonreducing terminal GalNAc(4SO 4 ) residue to the internal GalNAc(4SO 4 ) residue was 0.011 for CS-A and 0.021 for DS. When the chondroitinase ACII digests of CS-C was analyzed, the peak of GalNAc(4SO 4 ) was not detected under the same chromatographic conditions (data not shown). As the proportions of ⌬Di-4S in the total unsaturated disaccharides were 0.74 and 0.90 for CS-A and DS, respectively (29), molar ratios of GalNAc(4SO 4 ) to the total unsaturated disaccharide became 0.008 for CS-A and 0.019 for DS. These analytical data indicate that the contents of both internal and nonreducing terminal GalNAc(4SO 4 ) residue of DS were higher than those of CS-A; nevertheless, DS showed lower acceptor activity than CS-A as shown in Table II, suggesting that iduronic acid residues contained in DS may inhibit human GalNAc4S-6ST activity.
The recombinant human GalNAc4S-6ST transferred sulfate to the 4-sulfated oligosaccharides efficiently (Table II). To determine the position of the sulfate group transferred to the 4-sulfated oligosaccharides, the 35 S-labeled trisaccharide and pentasaccharide were digested with chondroitinase ACII and applied to SAX-HPLC (Fig. 6). In both the 35 S-labeled oligosaccharides, most of the radioactivity appeared at the position of GalNAc(4,6-bisSO 4 ) (Fig. 6, A and C). To establish the position to which 35 SO 4 was transferred, we digested the 35 S-labeled trisaccharide and pentasaccharide with chondro-6-sulfatase after digestion with chondroitinase ACII, and subjected it to SAX-HPLC. The radioactivity of GalNAc(4,6-bisSO 4 ) disappeared and was shifted to the position of inorganic sulfate (Fig.  6, B and D). These results indicate that human GalNAc4S-6ST transferred sulfate to position 6 of nonreducing terminal GalNAc(4SO 4 ) residues, but not to the reducing terminal and internal GalNAc(4SO 4 ) residues.

FIG. 5. Detection of nonreducing terminal GalNAc(4SO 4 ) in CS-A and DS by SAX-HPLC. CS-A (B)
and DS (C) were digested with chondroitinase ACII and chondroitinase ABC, respectively, and 500 nmol of the digests (as galactosamine) were applied to SAX-HPLC. The eluate was monitored with absorption at 210 nm. Elution profile of the standard materials were shown in A. Numbers above the peaks of the standard materials indicate: 1, ⌬Di-0S; 2, GalNAc(6SO 4 ); 3, GalNAc(4SO 4 ); 4, ⌬Di-6S; 5, ⌬Di-4S. Small peaks whose retention times were not agreed with those of the standards were not identified.

DISCUSSION
We have identified the human GalNAc4S-6ST cDNA for the first time. The strategy for the identification of the human GalNAc4S-6ST cDNA consisted of: 1) determination of amino acid sequences of the peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of the squid DNA by PCR using squid poly(A) ϩ RNA as a template and degenerate oligonucleotides as primers, and 3) FASTA search by the amino acid sequence deduced from the squid DNA fragment. Different lines of evidence indicated that the identified cDNA corresponds to the human counterpart of squid GalNAc4S-6ST previously purified from the squid cartilage: (a) the predicted amino acid sequence of the protein showed relatively high homology to the sequence deduced from the squid DNA; (b) the four of five peptides derived from the purified squid GalNAc4S-6ST nearly matched the human protein; (c) the expressed protein in COS-7 cells catalyzed the transfer of sulfate to position 6 of GalNAc(4SO 4 ) residues of CS-A and DS; (d) as observed in the purified squid enzyme, human GalNAc4S-6ST absolutely requires the presence of 4-sulfate moiety on GalNAc residue for its activity, because nonsulfated oligosaccharides did not serve as acceptor for human GalNAc4S-6ST; and (e) the predicted protein contained five potential N-linked glycosylation sites, which fits with the observation that the purified squid GalNAc4S-6ST is an N-linked glycoprotein (29). The predicted protein showed low but significant homology to 3O-ST family (41,42). Comparison of the sequence of human GalNAc4S-6ST with human 3O-ST-3A revealed ϳ25% identity and ϳ40% similarity at the amino acid level (Fig. 7). Relatively high homology was observed in the putative PAPS binding domains (5Ј-PSB and 3Ј-PB) and the carboxyl-terminal region.
Although both human and squid GalNAc4S-6ST transfer sulfate to position 6 of GalNAc(4SO 4 ) residue, a clear difference in the recognition of the targeted sugar residue is present between human and squid GalNAc4S-6ST. When CS-A was used as the acceptor, the squid GalNAc4S-6ST transferred sulfate mainly to position 6 of GalNAc(4SO 4 ) residues in the repeating disaccharide units. In contrast, human GalNAc4S-6ST transferred sulfate to position 6 of GalNAc(4SO 4 ) residue located at the nonreducing terminal and repeating disaccharide units at a nearly equal rate. The preference of human GalNAc4S-6ST to the nonreducing terminal GalNAc(4SO 4 ) residues was much more evident when DS was used as the acceptor. The observed difference in the substrate specificity between human and squid GalNAc4S-6ST may be related not only to the difference in the amino acid sequence but also to the fact that the squid enzyme is the natively expressed enzyme, whereas the human enzyme is a recombinantly expressed protein. The rate of sulfation of DS by human GalNAc4S-6ST was much lower than the rate of sulfation of CS-A (Table II). The relatively poor acceptor activity of DS may be the result of the presence of iduronic acid residue but of the lower concentration of nonreducing terminal GalNAc(4SO 4 ), because the proportion of GalNAc(4SO 4 ) residue in the total repeating disaccharide units of DS was larger than that of CS-A. CS-C hardly served as an acceptor. The inability of CS-C as the acceptor may be attributable not only to the very low contents of nonreducing terminal GalNAc(4SO 4 ) residue but also to the higher contents of GalNAc(6SO 4 ) residue; GalNAc(6SO 4 ) residue might inhibit the sulfation of the adjacent GalNAc(4SO 4 ) residue. Further works using oligosaccharides with the defined structures are required to reveal the effects of GalNAc(6SO 4 ) residue on the sulfation of both nonreducing terminal and internal GalNAc(4SO 4 ).
Human GalNAc4S-6ST transferred sulfate to the 4-sulfated oligosaccharides more efficiently than CS-A. Human GalNAc4S-6ST transferred sulfate predominantly to position 6 of nonreducing terminal GalNAc(4SO 4 ) residues when the 4-sulfated trisaccharide and 4-sulfated pentasaccharide were used as acceptors. Human GalNAc4S-6ST failed to transfer sulfate to the reducing terminal and internal GalNAc(4SO 4 ) residues of these oligosaccharides. Unlike these oligosaccharides, the transfer of sulfate to CS-A occurred at the internal GalNAc(4SO 4 ) residues. Such discrepancy in the acceptor specificity of human GalNAc4S-6ST might be accounted for by a unique conformation of CS-A that is not feasible for the oligosaccharides.
Vertebral sulfotransferases capable of producing GalNAc(4,6-bisSO 4 ) residues have been reported in quail oviduct (27), and human serum (28). These sulfotransferases mainly catalyzed sulfation of position 6 of nonreducing terminal GalNAc(4SO 4 ) residues. Such specificity is similar to that of human GalNAc4S-6ST. Human GalNAc4S-6ST may thus be involved in the formation of the nonreducing terminal GalNAc(4,6-bisSO 4 ) residues found in chondroitin sulfate chains attached to thrombomodulin (3) or aggrecan (23). Human GalNAc4S-6ST may also be involved in the synthesis of CS-E contained in the granules of mast cells, because this enzyme could catalyze the sulfation of the internal GalNAc(4SO 4 ) residues when CS-A was used as acceptor. However, the specificity of human GalNAc4S-6ST appears to be not suitable for the synthesis of the glycosaminoglycan containing IdoA␣1-3GalNAc(4,6-bisSO 4 ) unit, which was found in rat glomeruli (43) and rat mesangial cells (44), because human GalNAc4S-6ST transfers sulfate mainly to the nonreducing terminal GalNAc(4SO 4 ) residues when DS was used as acceptor. Inoue et al. (45) reported that the substrate specificity of the partially purified GalNAc4S-6ST from human serum depended on the pH of the reaction mixtures; the rate of sulfation of the internal GalNAc(4SO 4 ) residues increased as the pH was lowered. It remains to be determined whether the activity of human GalNAc4S-6ST toward the internal GalNAc(4SO 4 ) may also depend on the pH of the reaction mixtures. Alternatively, an isoform of human GalNAc4S-6ST that transfers sulfate mainly to the internal GalNAc(4SO 4 ) residue might be present in the mast cells.
Surprisingly, the nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell RAG-associated gene. The RAG1 and RAG2 play an important role in V(D)J recombination (46). hBRAG was cloned from Nalm-6 pre-B cell library as a cDNA that coexpressed closely with RAG1 mRNA (30). hBRAG gene was mapped to 10q26 (30). The expression pattern of hBRAG in human pro-B, pre-B, and mature B cell lines was closely related to that of RAG1. In human tissues, hBRAG is expressed in B cell-enriched tissues such as bone marrow and tonsil, but is not expressed in fetal or adult thymus. The product of hBRAG was shown to potentially involve in B cell-specific regulation of the expression of RAG1, because stable transfection of the complete hBRAG cDNA into a low RAG-expressing B-cell variant increased levels of RAG1 transcripts, but not in a nonlymphoid cell line (30). Immunoblotting and immunoprecipitation with the antibody raised against hBRAG protein demonstrated that hBRAG protein is expressed at both the cell surface and intracellular location of B cells as a membraneintegrated glycoprotein (47). The nucleotide sequence of the coding region of hBRAG is 99% identical to that of human GalNAc4S-6ST, but amino acid sequences of these proteins are rather different from each other. Missense mutations are observed in the codons corresponding to amino acid residues 180, 239, 240, and 241. Deletion of a nucleotide corresponding to amino acid residue181 results in a frameshift mutation. The reading frame is corrected by the insertion of a nucleotide at the codon corresponding to amino acid residue 223. Additional deletion of a nucleotide corresponding to amino acid residue 489 results in the appearance of a stop codon corresponding to amino acid residue 503. The hBRAG protein may not be a counterpart of the squid GalNAc4S-6ST because peptide 4 derived from the squid GalNAc4S-6ST did not match the hBRAG protein. Despite such alterations in the amino acid sequence, hBRAG protein still contains the PAPS binding domains that are characteristic to most of sulfotransferases so far cloned, suggesting that hBRAG protein may be a novel sulfotransferase whose substrate specificity is related to human GalNAc4S-6ST. It remains to be studied in future works whether formation of GalNAc(4,6-bisSO 4 ) residue might be involved in the regulation of the expression of RAG in B cells. Another hBRAG cDNA (accession no. AB025341) was also isolated from a human fetal brain library, and the hBRAG gene was shown to consist of seven exons and six introns (24). The nucleotide sequence of the hBRAG cDNA isolated from the human fetal brain library was strictly the same as the sequence of the cDNA that was identified as human GalNAc4S-6ST in this paper. The observed difference in the nucleotide sequence between hBRAG isolated from Nalm-6 pre-B cell and human GalNAc4S-6ST suggests that tissue-specific variants of GalNAc4S-6ST might be present.