Molecular Cloning and Characterization of GalNAc 4-Sulfotransferase Expressed in Human Pituitary Gland*

We have previously cloned chondroitin-4-sulfotrans-ferase (C4ST) cDNA from mouse brain. In this paper, we report cloning and characterization of GalNAc 4-sulfo-transferase (GalNAc4ST), which transfers sulfate to position 4 of the nonreducing terminal GalNAc residue. The obtained cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 424 amino acid residues. Identity of the amino acid sequence between GalNAc4ST and human C4ST was 30%. When the cDNA was transfected in COS-7 cells, sulfotransferase activity toward carbonic anhydrase VI was overexpressed but no sulfotransferase activity toward chondroitin or desulfated dermatan sulfate was increased over the control. Sulfation of carbonic anhydrase VI by the recombinant GalNAc4ST occurred at position 4 of the GalNAc residue of N -linked oligosaccharides. The recombinant GalNAc4ST transferred sulfate to position 4 of GalNAc residue of p -nitro-phenyl GalNAc, indicating that this sulfotransferase transfers sulfate to position 4 at the nonreducing terminal GalNAc residue. Dot blot analysis showed that the message of GalNAc4ST was expressed strongly in the human pituitary, suggesting that the cloned GalNAc4ST

Sulfated sugar chains are found not only in glycosaminoglycans but also in oligosaccharides of glycoproteins and glycolipids (1). Sulfate moieties attached to the sugar residues of glycosaminoglycans and oligosaccharides play key roles in various molecular and cellular interactions: binding of FGF2 to heparan sulfate (2,3); interaction of L-selectin on the lymphocytes with L-selectin ligands on the endothelial cells of high endothelial venule (4 -10); binding of HNK-1 epitope to the sulfo-glucuronyl carbohydrate-binding protein (11); and rapid clearance of a pituitary glycoprotein hormone, lutropin, mediated by the interaction with a hepatic reticuloendothelial cell receptor (12,13). Various sulfotransferases involved in the sulfation of glycosaminoglycans (14) and oligosaccharides (15)(16)(17) have been cloned. We have purified and cloned chondroitin-6-sulfotransferase (C6ST) 1 (18,19) and chondroitin-4-sulfotransferase (C4ST) (20,21), which are involved in the sulfation of position 6 and position 4, respectively, of GalNAc residues of chondroitin. C6ST also transfers sulfate to position 6 of Gal residue of keratan sulfate and sialyl N-acetyllactosamine oligosaccharides (22,23). We have cloned keratan sulfate Gal-6-sulfotransferase using the homology with C6ST. Keratan sulfate Gal6ST transfers sulfate to position 6 of the Gal residue of keratan sulfate and sialyl N-acetyllactosamine oligosaccharides but not to GalNAc residue of chondroitin (24,25). Several GlcNAc-6sulfotransferases, which are involved in the synthesis of 6-sulfo-sialyl Lewis x, have been cloned from the family genes including C6ST and keratan sulfate Gal6ST (9,10,26). On the other hand, C4ST showed significant homology with HNK-1 sulfotransferase (21,27), which transfers sulfate to position 3 of nonreducing terminal GlcA residue and is responsible for the synthesis of the HNK-1 epitope (16,17). These observations suggest that, in some cases, sulfotransferases involved in the sulfation of glycosaminoglycans and sulfotransferases involved in the sulfation of oligosaccharides of glycoproteins may be included in a common gene family.
Nonreducing terminal GalNAc 4-sulfate residue is present in oligosaccharides attached to pituitary glycoprotein hormones (lutropin, follitropin, and thyrotropin) (28 -30), pro-opiomelanocortin (31), and carbonic anhydrase VI of submaxillary gland (32), and was shown to play an important role in a pulsatile appearance of lutropin in the blood through the binding to the hapatic receptor for the sulfated GalNAc residue (12,33). Gal-NAc 4-sulfotransferase (GalNAc4ST) that transfers sulfate to 1 The abbreviations used are: C6ST, chondroitin-6-sulfotransferase; C4ST, chondroitin-4-sulfotransferase; GalNAc4ST, GalNAc 4-sulfotransferase; PAPS, 3Ј-phosphoadenosine 5Ј-phosphosulfate; GlcA, Dglucuronic acid; IdoA, L-iduronic acid; ⌬Di-0S, 2-acetamide-2-deoxy- the nonreducing terminal GalNAc residue attached to the Nlinked oligosaccharides of lutropin was found in the pituitary (34) and submaxillary gland (32), and was purified from the bovine submaxillary gland (35). Since both GalNAc4ST and C4ST transfer sulfate to position 4 of GalNAc residue, it is possible that GalNAc4ST and C4ST may belong to the same gene family as discussed above. On the basis of these considerations, we tried to find GalNAc4ST cDNA among expressed sequence-tagged cDNA clones showing homology with C4ST. One of these clones, which was expressed strongly in the pituitary, was found to encode a sulfotransferase capable of sulfating oligosaccharides of carbonic anhydrase VI. Product analysis showed that this sulfotransferase transferred sulfate to position 4 of the nonreducing terminal GalNAc residue.
Preparation of Carbonic Anhydrase VI from Bovine Submaxillary Gland-Carbonic anhydrase VI was purified from bovine submaxillary gland as described previously (32). All operations were carried out at 4°C. 200 g of the freshly excised glands, which were obtained from a local slaughterhouse under the help of a veterinary, Dr. A. Mabuchi, were put through a meat grinder and homogenized by a Polytron homogenizer in 1 liter of 50 mM sodium phosphate buffer, pH 7.4, 1 mM EDTA. The homogenate was centrifuged at 10,000 ϫ g, and the supernatant was filtered through two layers of cotton cloth and then precipitated with an equal volume of a saturated ammonium sulfate solution for 1 h. The solution was centrifuged at 10,000 ϫ g, and the precipitate was resuspended with 60 ml of 0.1 M NH 4 HCO 3 and dialyzed against 4 changes of 1.5 liter of 0.1 M NH 4 HCO 3 . After centrifuging the dialysate at 100,000 ϫ g for 60 min, one-half of the solution (50 ml) was passed over 5 ml of p-aminomethylbenzene sulfonamide-agarose (Sigma) followed by washing with 150 ml each of 0.1 M NH 4 HCO 3 and 0.2 M NaI in 0.1 M NH 4 HCO 3 . The column was eluted in 40 ml of 0.4 M NaN 3 in 0.1 M NH 4 HCO 3 , and the fractions containing protein and carbonic anhydrase activity were pooled and dialyzed against 300 ml of 25 mM Tris-HCl, pH 7.4. This affinity chromatography was repeated once. The dialysate was bound to a 15-ml DEAE-Sephacel column equilibrated with 25 mM Tris-HCl, pH 7.4, followed by washing in 200 ml of 50 mM NaCl in 25 mM Tris-HCl, pH 7.4, and elution in 100 ml of 200 mM NaCl in 25 mM Tris-HCl, pH 7.4. The fractions containing protein and carbonic anhydrase activity were pooled and dialyzed against 25 mM Tris-HCl, pH 7.4. Carbonic anhydrase activity was determined by the method using phenol red (40). Through the purification, 20 mg of carbonic anhydrase was obtained. On SDS-PAGE, the purified carbonic anhydrase VI showed a single protein band of 41 kDa before N-glycosidase F digestion and 35 kDa after N-glycosidase F digestion (Fig. 4).
Polymerase Chain Reaction and Preparation of a Probe for Screening-When the sequence of mouse C4ST was used for the homology search, we found a human expressed sequence-tagged cDNA clone (accession number AC005615). Examination of the sequence of the cDNA indicated the presence of the nucleotide sequences corresponding to putative PAPS binding motifs (5Ј-PSB and 3Ј-PB) found in every sulfotransferases; therefore, we predicted that this cDNA might encode a novel sulfotransferase with the substrate specificity similar to that of C4ST. We designed oligonucleotide primers for PCR from the sequence of the clone to amplify a DNA fragment, which was used as a probe for screening cDNA library. The 5Ј and 3Ј primers were GACCGCCAGGG-TATCTTGCA and GAGTGCCGGTCCTTGAACCG, respectively. The PCR reaction was carried out in a final volume of 50 l containing 50 pmol each of the oligonucleotide primers, 1 l of human brain cDNA solution (OriGene Technologies), 0.2 mM each of four deoxynucleoside triphosphates, 1.5 unit of AmpliTaq polymerase (PerkinElmer Life Sciences). Amplification was carried out by 40 cycles of 94°C for 45 s, 44°C for 1.5 min, and 72°C for 1 min. Reaction products were subjected to electrophoresis and the amplified DNA band (416 base pairs) was recovered from the gel. The radioactive probe for screening of the cDNA library was prepared from the PCR product by the random oligonucleotide-primed labeling method (41) using [␣-32 P]dCTP (Amersham Pharmacia Biotech) and a DNA random labeling kit (Takara Shuzo).
Screening of gt 11 Library-Approximately 4 ϫ 10 5 plaques from the human fetal brain cDNA library (CLONTECH) were screened. Hybond N ϩ nylon membrane (Amersham Pharmacia Biotech) replicas of the plaques from the gt 11 cDNA library were fixed by the alkali fixation method recommended by the manufacturer, prehybridized in a solution containing 50% formamide, 5 ϫ SSPE, 5 ϫ Denhardt's solution, 0.5% SDS, 0.04 mg/ml denatured salmon sperm DNA for 3.5 h at 42°C. Hybridization was carried out in the same buffer containing 32 P-labeled probe for 16 h at 42°C. The filters were washed at 55°C in 1 ϫ SSPE, 0.1% SDS, and subsequently in 0.1 ϫ SSPE, 0.1% SDS, and positive clones were detected by autoradiography.
DNA Sequence Analysis-DNA from gt 11 positive clones were isolated and cut with EcoRI, which excised the cDNA insert. The fragments were inserted into pBluescript II vector (Stratagene). The complete nucleotide sequence was determined by the dideoxy chain termination method using a DNA sequencer (Applied Biosystem Model 373A). DNA sequences were compiled and analyzed using the MacVector computer programs (Oxford Molecular Group PLC).
Construction of pFLAGGalNAc4ST and Transient Expression of GalNAc4ST cDNA in COS-7 Cells-A DNA fragment which codes for full open reading frame was amplified by PCR using human GalNAc4ST cDNA as a template. The 5Ј and 3Ј primers were CG-CAAGCTTATGACCCTGCGACCTGGAACAATG and CAGGAATTCT-CAGAGCCCTGTTGCTCCCAGGAT, respectively. The PCR reaction was carried out by 40 cycles of 94°C for 45 s, 57°C for 1.5 min, and 72°C for 1 min. The PCR product was digested with EcoRI and HindIII, and subcloned into these sites of pFLAG-CMV-2 plasmid (Kodak, New Haven, CT). COS-7 cells (obtained from Riken Cell Bank, Tsukuba, Japan) were plated in 100-mm culture dishes at a density of 8 ϫ 10 5 cells/dish. Volume of the medium was 10 ml. The medium used was Dulbecco's modified Eagle's medium containing penicillin (100 units/ ml), streptomycin (50 g/ml), and 10% fetal bovine serum (Life Technologies, Inc.), and cells were grown at 37°C in 5% CO 2 , 95% air. When the cell density reached 3 ϫ 10 6 cells/dish (48 h after plating), COS-7 cells were transfected with pFLAGGalNAc4ST, a recombinant plasmid containing the GalNAc4ST cDNA in pFLAG-CMV-2, or pFLAG-CMV-2. The transfection was performed using the DEAE-dextran method (42). 5 ml of the prewarmed Dulbecco's modified Eagle's medium containing 10% Nu-serum (Collaborative Biomedical Products) was mixed with 0.2 ml of phosphate-buffered saline containing 10 mg/ml DEAE-dextran plus 2.5 mM chloroquine solution. 15 g of the recombinant plasmid was mixed with the solution, and the mixture was added to the cells. The cells were incubated for 4 h in a CO 2 incubator. The medium was then replaced with 5 ml of 10% dimethyl sulfoxide in phosphate-buffered saline. After the cells were left at room temperature for 2 min, the dimethyl sulfoxide solution was aspirated and 25 ml of Dulbecco's modified Eagle's medium containing penicillin (100 units/ml), streptomycin (50 g/ml), and 10% fetal bovine serum was added. After incubation for 60 -65 h, the cells were washed with Dulbecco's modified Eagle's medium alone, and the recombinant protein produced was extracted from the cells with a buffer containing 10 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 10 mM MgCl 2 , 2 mM CaCl 2 , 0.5% Triton X-100, 20% glycerol by gentle shaking on a rotatory shaker for 30 min at 4°C. The extracts were centrifuged at 10,000 ϫ g for 10 min. The supernatant fraction was used for the experiments on the recombinant GalNAc4ST.
Assay of C4ST Activity-C4ST activity was assayed by the method described previously (20). The standard reaction mixture contained 50 mM imidazole-HCl, pH 6.8, 0.0025% protamine chloride, 2 mM dithiothreitol, 25 nmol (as glucuronic acid) chondroitin, 50 pmol of [ 35 S]PAPS (about 5.0 ϫ 10 5 cpm), and enzyme in a final volume of 50 l. For determining the activity toward desulfated dermatan sulfate, chon-droitin was replaced with 25 nmol (as galactosamine) of desulfated dermatan sulfate and the amount of protamine chloride was increased to 0.02%. 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 and radioactivity was determined. For determining the incorporation into position 4 and position 6 of GalNAc residues, 35 S-labeled chondroitin and 35 S-labeled desulfated dermatan sulfate were digested with chondroitinase ACII and chondroitinase ABC, respectively. The resulting unsaturated disaccharides (⌬Di-4S and ⌬Di-6S) were separated with paper chromatography, and their radioactivities were measured.
Assay of GalNAc4ST Activity-GalNAc4ST activity was assayed using carbonic anhydrase VI as an acceptor by the method described previously (34) with slight modification. The standard reaction mixture contained 15 mM imidazole-HCl, pH 7.2, 6 mM Mg(CH 3 COO) 2 , 40 mM 2-mercaptoethanol, 1% Triton X-100, 10 mM NaF, 0.1 mM 5Ј-AMP, 13% glycerol, 10 g of the purified carbonic anhydrase VI, 50 pmol of [ 35 S]PAPS (about 5.0 ϫ 10 5 cpm), and enzyme in a final volume of 50 l. The reaction mixtures were incubated at 28°C for 2 h. After the reaction was over, the reaction mixtures were placed on an ice bath and injected into a Fast Desalting column as described previously and radioactivity of the void fraction was determined. Under the assay conditions, the incorporation of 35 SO 4 into carbonic anhydrase VI proceeded linearly up to 2 h. To determine the sulfation of pNP-GalNAc, carbonic anhydrase VI was replaced with 25 nmol of pNP-GalNAc. The reaction was stopped by adding 30 l of 0.1 M HCl and the mixtures were incubated at 37°C for 60 min to degrade excess amounts of [ 35 S]PAPS. After the mixtures were spotted on Toyo No. 51A filter paper, the filter paper was developed with a solvent described below until the solvent front reached the edge of the paper. The dried paper strips were cut into 1.25-cm segments, which were analyzed for radioactivity by liquid scintillation counting.
Dot blot hybridization-Human Multiple Tissue Expression Array was prehybridized in ExpresHyb solution (CLONTECH) at 68°C. Hybridization was carried out in the same solution containing 32 P-labeled probe for 1 h at 68°C. The radioactive probe was prepared from the cDNA fragment excised from the pBluescript II plasmid with EcoRI by the random oligonucleotide-primed labeling method using [␣-32 P]dCTP and a DNA random labeling kit (Takara Shuzo). The filters were washed at room temperature in 2 ϫ SSC, 0.05% SDS, and subsequently in 0.1 ϫ SSC, 0.1% SDS at 50°C. The membrane was exposed to x-ray film at Ϫ80°C with an intensifying screen.
SDS-Polyacrylamide Gel Electrophoresis-Polyacrylamide gel electrophoresis of proteins in SDS was carried out on 10% polyacrylamide gels as described (43). Protein bands were detected by Coomasie Brilliant Blue. 35 S radioactivity was detected by autoradiography after the gel was dried. Assay of Protein-Protein was determined by the method of Bradford using bovine serum albumin as a standard (44). Protein assay reagent was obtained from Bio-Rad.
Digestion of the Carbonic Anhydrase VI with N-Glycosidase F-The 35 S-labeled carbonic anhydrase VI was precipitated with 10% trichloroacetic acid. The precipitates were washed with acetone and digested with recombinant N-glycosidase F (Roche Molecular Biochemicals) by the methods recommended by the manufacturer. After digestion, the protein was precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE. Oligosacharides released by N-glycosidase F digestion were recovered from the supernatant of 10% trichloroacetic acid.
Superdex 30 Chromatography, Paper Electrophoresis, Paper 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 1 ml/min. One-ml fractions were collected. Paper electrophoresis was carried out on Whatman No. 3 paper (2.5 cm ϫ 57 cm) in pyridine/acetic acid/water (1:10: 400, by volume, pH 4) at 30 V/cm for 40 min. Paper chromatography was performed on Toyo No. 51A paper (20 ϫ 50 cm) using a solvent system, 1-butanol, acetic acid, 1 M NH 3 (2:3:1, by volume). The dried paper strips after paper electrophoresis or paper chromatography were cut into 1.25-cm segments, which were analyzed for radioactivity by liquid scintillation counting. Separation of GalNAc(4SO 4 ) was 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 indicted in Fig. 5. Fractions (0.5 ml) were collected at a flow rate of 1 ml/min and a column temperature of 40°C.

cDNA and Predicted Protein Sequence of the GalNAc4ST-
When approximately 4 ϫ 10 5 plaques of a human fetal brain cDNA library were screened using a probe, which was prepared by PCR using human brain cDNA as a template and primer oligonucleotides designed from the sequence of a human expressed sequence-tagged cDNA clone (accession number AC005615), two cDNA clones (2.2 and 1.3 kilobase pairs) were isolated. One of these clones (2.2 kilobase pairs) was found to contain whole open reading frame. The nucleotide sequence of the GalNAc4ST cDNA and the predicted amino acid sequence are shown in Fig. 1A. A single open reading frame predicts a protein of 424 amino acid residues with four potential N-linked glycosylation sites. Putative PAPS-binding domains (5Ј-PSB and 3Ј-PB) were present. The presumptive polyadenylation signal was found before the poly(A) sequence. To determine the location of any transmembrane domain, a hydropathy plot was generated from the translated sequence. Analysis of the plot revealed one prominent hydrophobic segment in the aminoterminal region, 22 residues in length, that extends from amino acid residues 10 to 31 (Fig. 1B).
Comparison of the coding sequence of human GalNAc4ST with that of human C4ST (45) has revealed that there are 30% identity on the amino acid level (Fig. 2). Homology in the amino acid sequence between the two proteins was observed in the carboxyl-terminal side of the molecules. Especially, amino acid sequences of 5Ј-PSB and 3Ј-PB were well conserved. Homology of the N-terminal region was rather poor.
Expression of GalNAc4ST cDNA in COS-7 Cells-COS-7 cells were transfected with the pFLAGGalNAc4ST, a recombinant plasmid containing the isolated cDNA in the mammalian  and 2), and the radioactivity was detected by autoradiography (lanes 3  and 4). Molecular size standards were the following: bovine serum albumin (66 kDa), egg albumin (45 kDa), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (36 kDa), and bovine erythrocyte carbonic anhydrase (29 kDa). expression vector pFLAG-CMV-2. The transfected cells were extracted with a buffer containing 0.5% Triton X-100 and centrifuged. Activities of sulfotransferase was determined using chondroitin, desulfated dermatan sulfate, or carbonic anhydrase VI as acceptors. Control experiments with vector alone were also done. As shown in Fig. 3, more than 10-fold overexpression of the sulfotransferase activity was observed when carbonic anhydrase VI was used as the acceptor. In contrast, no sulfotransferase activity was overexpressed when chondroitin or desulfated dermatan sulfate was used as the acceptor. Sulfotransferase activity toward carbonic anhydrase VI was not overexpressed when COS-7 cells were transfected with human C4ST cDNA (data not shown).
Analysis of Sulfated Carbonic Anhydrase VI-When 35 S-labeled carbonic anhydrase VI was separated with SDS-PAGE, the radioactivity was coincided with the protein band visual-ized with Coomasie Blue (Fig. 4, lane 1 and 3) The radioactivity, however, was completely removed after N-glycosidase F digestion (Fig. 4, lane 2 and 4), indicating that 35 SO 4 was transferred to N-linked oligosaccharides of carbonic anhydrase VI. The 35 S-labeled N-linked oligosaccharides released from the 35 S-labeled carbonic anhydrase VI with N-glycosidase F digestion were isolated by Superdex 30 chromatography (Fig.  5A). The 35 S-labeled oligosaccharide fractions were hardly obtained when carbonic anhydrase VI was incubated with the extracts from COS-7 cells transfected with vector alone (control extract) (open circle in Fig. 5A). The 35 S-labeled oligosaccharides were subjected to mild acid hydrolysis (40 mM HCl, 100°C, 120 min) and separated with the Superdex 30 column again (Fig. 5B). The 35 S radioactivity was detected in the position of GalNAc(4SO 4 ), inorganic sulfate and larger molecules, which were thought to be partially degraded oligosaccharides. The fractions corresponding to GalNAc(4SO 4 ) and inorganic sulfate (indicated by a horizontal bar in Fig. 5B) were combined. After removal of inorganic sulfate by paper electrophoresis, the materials which behaved together with GalNAc(4SO 4 ) in both Superdex 30 chromatography and paper electrophoresis were separated with SAX-HPLC (Fig. 5C). The 35 S radioactiv-  a horizontal bar) was eluted from the paper and used for further analysis. B, peak 2 fraction from A was separated by paper electrophoresis. Peak 3 (indicated by a horizontal bar) was eluted from the paper and subjected to mild acid hydrolysis (40 mM HCl, 100°C, 60 min). C, after the mild acid hydrolysis, peak 3 from B was separated by paper electrophoresis. Peak 6 represents inorganic sulfate. Peak 5 (indicated by a horizontal bar), which was observed only when pNP-GalNAc was incubated with recombinant GalNAc4ST, was pooled and used for further analysis. D, peak 5 fraction from C was separated by paper chromatography for 20 h. The arrows indicate the position of standard sugars detected by silver nitrate staining: a, ⌬Di-diS E ; b, GalNAc(4, 6-bisSO 4 ); c, ⌬Di-6S; d, ⌬Di-4S; e, GalNAc(6SO 4 ); f, GalNAc(3SO 4 ); and g, GalNAc(4SO 4 ).

FIG. 5. Identification of [ 35 S]GalNAc(4SO 4 ) in the mild acid hydrolysate of 35 S-labeled N-linked oligosaccharides released from 35 S-labeled carbonic anhydrase VI by N-glycosidase F digestion. A, carbonic anhydrase VI was incubated with the recombinant
ity was exclusively eluted at the position of GalNAc(4SO 4 ), and was clearly separated from GalNAc(6SO 4 ), GalNAc(3SO 4 ), GlcNAc(6SO 4 ), and GlcNAc(3SO 4 ). These observations clearly indicate that sulfate was transferred to position 4 of GalNAc residue of the N-linked oligosaccharides attached to carbonic anhydrase VI.

Analysis of Sulfated p-Nitrophenyl GalNAc-It was reported
that N-linked oligosaccharides attached to carbonic anhydrase VI contained GalNAc␤1-4GlcNAc sequence at the nonreducing terminal (32); therefore, it is most likely that 35 SO 4 was transferred to GalNAc residue at the nonreducing terminal. To demonstrate that the recombinant GalNAc4ST could transfer sulfate to nonreducing terminal GalNAc residue, we tested the possibility that p-nitrophenyl-␤-D-GalNAc (pNP-GalNAc) could serve as acceptor for GalNAc4ST, since pNP-GalNAc was reported to inhibit GalNAc4ST activity (34). After pNP-GalNAc was incubated with the recombinant GalNAc4ST together with [ 35 S]PAPS, the reaction products were separated with paper chromatography. A radioactive peak migrating near the solvent front was observed (peak 2 in Fig. 6A). This peak was also observed when pNP-GalNAc was incubated with the control extract. The radioactive materials contained in peak 2 (indicated by a horizontal bar in Fig. 6A) were eluted from the paper and separated with paper electrophoresis (Fig. 6B). Two peaks (peaks 3 and 4 in Fig. 6B) were observed when pNP-GalNAc was incubated with the recombinant GalNAc4ST. The slower migrating peak (peak 3) was not observed when pNP-GalNAc was incubated with the control extract. When peak 3 in Fig. 6B (indicated by a horizontal bar) was eluted, subjected to mild acid hydrolysis (40 mM HCl, 100°C, 60 min) (46), and separated again with paper electrophoresis, a radioactive peak (peak 5 in Fig. 6C), which migrated slowly than inorganic sulfate (peak 6 in Fig. 6C), was observed when pNP-GalNAc was incubated with the recombinant GalNAc4ST. A small peak was observed slightly ahead of peak 5, but this peak was not examined further. Peak 5 was not detected at all when pNP-GalNAc was incubated with the control extract. The mild acid hydrolysis of peak 4 in Fig. 6B resulted in complete release of inorganic sulfate even when pNP-GalNAc was incubated with the recombinant GalNAc4ST (data not shown). When peak 5 was recovered and separated with paper chromatography, the 35 S radioactivity was detected in two peaks (Fig. 6D). One of the two peaks (peak 7 in Fig. 6D) migrated to the position of GalNAc(4SO 4 ) and was clearly separated from GalNAc(6SO 4 ) and GalNAc(3SO 4 ). The faster migrating peak (peak 8 in Fig.  6D) seemed to contain sulfated pNP-GalNAc which remained intact during the mild acid hydrolysis. These observations clearly indicate that GalNAc4ST transfers sulfate to position 4 of nonreducing terminal GalNAc residue. Both the recombinant GalNAc4ST and the control extracts catalyzed the formation of 35 S-labeled material that was degraded completely by the mild acid hydrolysis (peak 4 in Fig. 6B). Since this acid-  Fig. 6. A, the reaction mixtures were spotted on paper and developed until solvent front reached the paper edge (about 12 h). Peak 1 in A (indicated by a horizontal bar) was eluted from the paper and used for further analysis. B, peak 1 from A was separated by paper electrophoresis. Only one peak (peak 2) was obtained even when pNP-GlcNAc was incubated with the recombinant GalNAc4ST (closed circle). Peak 2 (indicated by a horizontal bar in B) was subjected to mild acid hydrolysis (40 mM HCl, 100°C, 60 min). C, after the mild acid hydrolysis, peak 2 from B was separated with paper electrophoresis. All the radioactivity migrated to the position of inorganic sulfate (peak 3).  ). B, the GalNAc4ST activity was determined as described under "Experimental Procedures" except that the concentration of 2-mercaptoethanol was varied. C, the GalNAc4ST activity was determined as described under "Experimental Procedures" except that various amounts of protamine chloride were added to the reaction mixtures. FIG. 9. Effect of the concentration of carbonic anhydrase VI on the activity of the recombinant GalNAc4ST. The GalNAc4ST activity was determined as described under "Experimental Procedures" except that the concentration of carbonic anhydrase VI was varied. The inset represents the double reciprocal plot, in which the concentration of carbonic anhydrase VI was calculated on the assumption that molecular weight of carbonic anhydrase VI is 41,000. labile 35 S-labeled material was formed when p-nitrophenol was used as acceptor, and was migrated together with p-nitrophenyl sulfate in paper chromatography and paper electrophoresis (data not shown), this material appears to be p-nitrophenyl sulfate. p-Nitrophenyl sulfate might be formed by the sulfation of contaminating p-nitrophenol in pNP-GalNAc with endogenous cytosol sulfotransferase. Unlike pNP-GalNAc, no sulfated GlcNAc was obtained when pNP-GlcNAc was used as acceptor, although acid-labile 35 S-labeled material was formed (Fig. 7). These results suggest that GalNAc4ST may not transfer sulfate to nonreducing terminal GlcNAc residue.
Properties of GalNAc4ST-The pH optimum for the recombinant GalNAc4ST was around 7.2 (Fig. 8A). The recombinant GalNAc4ST was stimulated with 2-mercaptoethanol (Fig. 8B) and protamine chloride (Fig. 8C). These properties were similar to those of the GalNAc4ST preparation from the bovine pituitary (34). The K m for carbonic anhydrase VI was 10 M on the assumption that molecular weight of the purified carbonic anhydrase VI is 41,000 (Fig. 9). This value is similar to the K m for GalNAc␤1-4GlcNAc␤1-2Man␣-O-(CH 2 ) 8 -COOCH 3 of the pituitary GalNAc4ST (34) .
Dot Blot Analysis-Dot blot analysis using Human Multiple Tissue Expression Array (CLONTECH) showed that GalNAc4ST was expressed in various brain tissues and pla-centa; the strongest expression was observed in the pituitary gland (Fig. 10). DISCUSSION We have cloned GalNAc4ST from a fetal brain library as a protein showing sequence homology with C4ST. GalNAc4ST shared several properties with C4ST: 1) both sulfotransferases were type II transmembrane proteins having four potential N-glycosylation sites. 2) Amino acid sequences of the putative PAPS-binding domains, especially 3Ј-PB, of these sulfotransferases were highly conserved. 3) 2-Mercaptoethanol and protamine chloride activated both sulfotransferases. 4) Both sulfotransferases transferred sulfate to position 4 of GalNAc residue. However, expression pattern in various human tissues were quite different; human C4ST was expressed strongly in peripheral blood leukocytes (27,45) and colorectal adenocarcinoma (45), whereas expression of GalNAc4ST was detected in the various brain-related tissues and placenta. The strongest expression of GalNAc4ST was observed in the pituitary gland, suggesting that the cloned GalNAc4ST might participate in the biosynthesis of nonreducing terminal GalNAc(4SO 4 ) residue found in N-linked oligosaccharides of pituitary hormones. As observed in C4ST, GalNAc4ST also contains Cys in the 5Ј-PSB domain, and was activated with 2-mercaptoethanol, suggesting that the Cys residue in 5Ј-PSB may be relevant to the stimulation of GalNAc4ST and C4ST by 2-mercaptoethanol.
Although both C4ST and GalNAc4ST transfer sulfate to position 4 of GalNAc residue, a clear difference in the recognition of the neighboring sugar residue was observed between these sulfotransferases. GalNAc residues in the repeating disaccharide units of chondroitin, GalNAc␤1-4GlcA, acted as acceptor for C4ST, but did not serve as acceptor for GalNAc4ST. On the other hand, GalNAc residues in the nonreducing terminal GalNAc␤1-4GlcNAc sequence present in N-linked oligosaccharides of carbonic anhydrase VI did not serve as acceptor for C4ST. It has been reported that isoforms of a glycosaminoglycan sulfotransferase transferred sulfate to the same position of the same sugar residue, but showed difference in the recognition of the structure of the neighboring sugar residue. Both 3O-ST-1 and 3O-ST-2 transferred sulfate to position 3 of GlcN(SO 4 ), but 3O-ST-1 required GlcA at the nonreducing side, whereas 3O-ST-2 required IdoA(2SO 4 ) or GlcA(2SO 4 ) (47, 48). HS6ST-1, -2, and -3 transferred sulfate to position 6 of Glc-N(SO 4 ) of heparan sulfate, but each isoform showed the different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer iduronosyl N-sulfoglucosamine unit, while HS6ST-2 had the different substrate preference depending upon the concentration of substrate and HS6ST-3 acted on either substrate (49). To understand the substrate specificity, it will be required to establish the three-dimensional interaction between each sulfotransferase and acceptor substrates.
Nonreducing terminal GalNAc(4SO 4 )␤1-4GlcNAc sequence found in pituitary hormones has been implicated in the pulsatile characteristic of the circulating hormone levels through binding to the receptor for sulfated GalNAc␤1-4GlcNAc termini expressed by hepatic endothelial cells and Kupffer cells (12,33). A pituitary sulfotransferase responsible for the 4-Osulfation of terminal GalNAc residue was characterized using GalNAc␤1-4GlcNAc␤1-2Man␣-O-(CH 2 ) 8 -COOCH 3 (GGnM-MCO) as an acceptor (34), and the sulfotransferase with the same substrate specificity as that of the pituitary sulfotransferase was purified from bovine submaxillary gland (35). The recombinant GalNAc4ST expressed in COS-7 cells from the cDNA shared several properties with the purified GalNAc4ST. The pH optimum of both the recombinant GalNAc4ST and the purified GalNAc4ST fell between 7.0 and 7.5, and both the sulfotransferases were activated with 2-mercaptoethanol and protamine chloride. In contrast, molecular size of the purified GalNAc4ST was quite different from that of the recombinant GalNAc4ST; molecular size of the purified GalNAc4ST was 128 kDa on SDS-PAGE (35), whereas molecular mass of the recombinant GalNAc4ST calculated from the cDNA was 48,831. Such a discrepancy in molecular size may be explained by a hypothesis that the purified GalNAc4ST might be present as a dimer. The protein deduced from the GalNAc4ST cDNA potentially bears four N-linked oligosaccharide chains (Fig. 1A). C4ST also contains four potential glycosylation sites (21), and the contents of N-linked oligosaccharide of the purified C4ST was estimated as 35% as judged from the decrease in molecular size after digestion with N-glycosidase F (20). If the content of N-linked oligosaccharides of the GalNAc4ST is nearly equal to that of C4ST, molecular size of the glycosylated form of the recombinant GalNAc4ST could be estimated as about 64 kDa. This value is just a half of the molecular size reported for the purified GalNAc4ST. Alternatively, GalNAc4ST expressed in the pituitary and GalNAc4ST present in the submaxillary gland may be quite distinct from each other.
Molecular size of the purified carbonic anhydrase VI was 41 kDa as judged from the mobility on SDS-PAGE (Fig. 4). This value seems to be slightly smaller than the molecular size previously reported (50). After digestion with N-glycosidase F, molecular size was decreased to 35 kDa, which is nearly the same as that previously reported; therefore, the difference in the molecular size of the intact carbonic anhydrase VI might be attributable to the heterogeneity in the glycosylation.
We found pNP-GalNAc served as an acceptor for the recombinant GalNAc4ST. pNP-GalNAc was reported to inhibit pituitary GalNAc4ST (34), but it has not been examined whether this material could serve as acceptor for the pituitary or submaxillary GalNAc4ST. Comparison of the hydropathy plot between GalNAc4ST and C4ST revealed that GalNAc4ST, but not C4ST, had a cluster of hydrophobic amino acid residues (Val 208 -Ala 216 ) near the 5Ј-PSB (Fig. 1). The presence of the hydrophobic region might contribute to the recognition of hydrophobic aglycon bound to the targeted GalNAc residue such as the p-nitrophenyl group or GlcNAc residue. We found that the recombinant GalNAc4ST failed to transfer sulfate to a undecasaccharide prepared from chondroitin by the digestion with hyaluronidase and ␤-glucuronidase (data not shown). The penultimating hydrophilic GlcA residue contained in the undecasaccharide might inhibit the recognition of nonreducing terminal GalNAc residue by GalNAc4ST. We previously found that C6ST-containing microsomal fraction of chick embryo chondrocytes catalyzed sulfation of position 6 of the GalNAc residue of pNP-GalNAc (46), but unlike the sulfation with GalNAc4ST, 6-sulfation by the microsomal fraction was markedly inhibited by the addition of detergent.
By BLAST search, we obtained human genomic clones located to chromosome 19q13.1 that contained identical sequence with the cDNA of GalNAc4ST. Genomic organization constructed from these genomic clones showed that there were at least four exons; start ATG codon and terminal TGA codon were found in the second exon and the fourth exon, respectively (Fig. 11). Nucleotide sequences of the exon-intron junctions fitted the consensus sequence except for 5Ј-terminal of the first