Molecular Cloning and Characterization of anN-Acetylglucosamine-6-O-sulfotransferase*

We isolated a cDNA clone encoding mouseN-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcβ1–3Galβ1–4GlcNAc. Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcα2–3Galβ1–4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galβ1–4(Fucα1–3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situhybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

Sulfation of carbohydrate chains is an important step in construction of most glycosaminoglycans (1) and is also observed in glycoproteins (2-7) and glycolipids (8). Several sulfotransferases catalyzing carbohydrate sulfation have been cloned recently, opening the new era in understanding biological functions and biosynthetic regulation of sulfated glycans (9 -15). However, the cloned sulfotransferases are those which transfer sulfate to galactose (10,15,16), glucuronic acid (14), iduronic acid (12), N-acetylgalactosamine (9), or to the hydroxyl (13) or amino groups (11) of glucosamine. No enzymes capable of transferring sulfate to hydroxyl groups of N-acetylglucosamine (GlcNAc) in glycoconjugates have been cloned to date.
Here, we describe molecular cloning and characterization of a GlcNAc-6-O-sulfotransferase. The localization of the transcript in high endothelial venules (HEV) 1 of mesenteric lymph nodes suggested that the enzyme is involved in synthesis of an L-selectin ligand.
The following glycolipids were generous gifts from Dr. Makoto Kiso, Faculty of Agriculture, Gifu University; sialyl Lewis X ceramide, NeuAc␣2-3Gal␤1-4(Fuc␣1-3)GlcNAc␤1-3Gal␤1-4Glc␤1-Cer; 6-sulfo * This work was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Science and Culture. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB011451.
§ Research Fellow of the Japanese Society for the Promotion of Science.
Isolation of GlcNAc-6-O-sulfotransferase cDNA-A mouse expressed sequence tag sequence (Genbank TM accession no. AA103962) with similarity to the catalytic portion of mouse chondroitin-6-sulfotransferase was amplified by the reverse transcription-PCR method using mouse day 13 embryo total RNA as a template. The sense primer 5Ј-GTCGTCGGACTGGTGGACGA-3Ј and the antisense primer 5Ј-CCCA-GAGCGTGGTAGTCTGC-3Ј were used for PCR amplification, which was carried out at 94°C for 3 min, with 35 cycles of 94°C for 0.5 min, 60°C for 1 min, and 72°C for 1 min. The PCR product (368 bp) was 32 P-labeled with a Megaprime TM DNA labeling system (Amersham Pharmacia Biotech) and used to screen the gt 11 mouse day 7 embryo cDNA library. Hybridization was carried out as described previously (9). DNA inserts were isolated from positive gt 11 clones by digestion with EcoRI and subcloned into pBluescript II SKϪ (STRATAGENE). Then, the nucleotide sequence was determined by the dideoxy chain termination method (29) using an Applied Biosystems automated sequencer.
Construction of Expression Vectors-A cDNA fragment encoding the open reading frame of GlcNAc-6-O-sulfotransferase was amplified by PCR using primers, 5Ј-ACGAATTCGGGATGAAGGTATTTCGCAGG-3Ј and 5Ј-ATGAATTCTCAAAGCCGGGGCTTCCTGAG-3Ј, and the cloned cDNA fragment as a template. PCR amplification was carried out at 94°C 3 min, with 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min in 5% (v/v) dimethyl sulfoxide. The PCR product including the open reading frame of GlcNAc-6-O-sulfotransferase (nucleotide num- bers 467-1921 in Fig. 1A) was digested with EcoRI and subcloned into the pcDNA3 expression vector (Invitrogen). A recombinant plasmid with the correct orientation, pcDNA3-GlcNAc6ST, was used for expression. The plasmid containing the fragment in the reverse orientation, pcDNA3-GlcNAc6STA, was used in control experiments. The previously cloned 1544-bp fragment of the mouse fucosyltransferase IV gene (30) was subcloned into the BamHI and EcoRI sites of the pcDNA3 expression vector as described above. The plasmid containing the insert in the correct orientation, pcDNA3-FucTIV, was used.
Transient Expression of GlcNAc-6-O-sulfotransferase cDNA-COS-7 cells (3 ϫ 10 6 cells/10-cm dish) were transfected with 15 g of relevant plasmids by the DEAE-dextran method (31). After 65 h of culture in Dulbecco's modified minimum essential medium containing 10% fetal calf serum, the cells were washed with phosphate-buffered saline, scraped off the dishes and homogenized with a Dounce homogenizer in 1.5 ml/dish of 0.25 M sucrose, 20 mM Tris-HCl, pH 7.2, and 0.5% Triton X-100. The homogenates were centrifuged at 10,000 ϫ g for 15 min, and the supernatant was saved as the extract. For FACS analysis, the transfected cells were cultured for 48 h, passed into 25-cm 2 culture flasks (3 ϫ 10 5 cells/flask), and further cultured for 36 h.
Assay of Sulfotransferase Activity to Various High Molecular Weight Substrates-Sulfotransferase activities were assayed using various glycosaminoglycans as described previously (10,32). When oligosaccharides were used as substrates, the reaction mixture contained 2.5 mmol of imidazole-HCl, pH 6.8, 0.5 mol of MnCl 2 , 0.1 mol of AMP, 1.0 mol of NaF, 25 nmol of oligosaccharides, 50 pmol of [ 35 S]PAPS (about 5 ϫ 10 5 cpm), and 5 l of the extracts from COS-7 cells in a final volume of 50 l. The reaction mixture was incubated at 30°C for 5 h, and the reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. 35 S-Labeled oligosaccharides were separated from 35 SO 4 and [ 35 S]PAPS by Superdex 30 gel chromatography, and the radioac-tivity was determined. When GlcNAc␤1-3Gal␤1-4GlcNAc was used as an acceptor, sulfotransferase reaction proceeded linearly up to 5 h under the assay conditions.
N-Deacetylation, Deamination, and NaBH 4 Reduction of the 35 S-Labeled Sulfated GlcNAc␤1-3Gal␤1-4GlcNAc-The 35 S-labeled sulfated GlcNAc␤1-3Gal␤1-4GlcNAc was prepared using the expressed GlcNAc-6-O-sulfotransferase (2.1 g as protein in the extract) as described above except that the concentration of [ 35 S]PAPS was increased to 6-fold and incubation was carried out for 25 h. The 35 S-labeled sulfated GlcNAc␤1-3Gal␤1-4GlcNAc eluted from the Superdex 30 column was lyophilized, purified by paper electrophoresis, and deacetylated with 70% hydrazine containing 1.0% hydrazine sulfate at 95°C for 30 h (33). The deacetylated materials were purified by Superdex 30 chromatography, subjected to deamination with nitrous acid at pH 4, and reduced by NaBH 4 (16). Finally the sample was dissolved in 60 l of water and subjected to paper chromatography.
Periodate Oxidation-The 35 S-labeled sulfated 2,5-anhydromannitol sample eluted after paper chromatography or 3 H-labeled (SO 4 -3)2,5anhydromannitol were lyophilized and dissolved in 20 l of 50 mM sodium acetate buffer, pH 4.0, containing 80 mM NaIO 4 . After 20 h of incubation at 4°C in the dark, 5 l of 20% ethylene glycol were added to reduce the remaining NaIO 4 . After standing for 1 h at room temperature, the samples were subjected to paper chromatography.
Northern and Genomic Southern Blot Analyses-Total RNA (20 g) was prepared from C57 BL/6J mouse tissues as described elsewhere (36). Genomic DNA (10 g) prepared from murine D3 embryonic stem cells (37) was digested for 4 h with appropriate restriction enzymes. The radioactive probe was the same as that used for screening of the mouse day 7 embryo cDNA library. The blots were washed at 55°C in 2ϫ SSPE, 0.1% SDS, and finally in 0.1ϫ SSPE, 0.1% SDS at 55°C. The membranes were exposed to a BAS-imaging plate and then the radioactivity on the membrane was determined with a BAS 2000 radioimage analyzer (Fuji Film).
In Situ Hybridization-Mesentric lymph nodes from C57 BL/6J mice were subjected to hematoxylin-eosin staining or in situ hybridization as described previously (38). As the GlcNAc-6-O-sulfotransferase probe, a 0.6-kbp PstI fragment of the cDNA (nucleotide numbers 962-1561 in

Overexpression of GlcNAc-6-O-sulfotransferase in COS-7 cells
Sulfotransferase activities in the extracts of the cells transfected with the pcDNA3-GlcNAc6ST (sense) or the pcDNA3-GlcNAc6STA (antisense) or no plasmids (none) were determined using GlcNAc␤1-3Gal␤1-4GlcNAc as a sulfate acceptor. The activity was calculated from the radioactivity contained in fraction numbers 85-89 on Superdex 30 chromatography (Fig. 3A). Values obtained in the absence of the acceptor (cf. open circles in Fig. 3A) were subtracted from the values of experimental runs (cf. closed circles in Fig. 3A).

Plasmids
Sulfotransferase activity a pmol/h/mg protein

Molecular
Cloning of a cDNA Homologous to Chondroitin-6sulfotransferase-We previously cloned a cDNA encoding mouse chondroitin-6-sulfotransferase (39). By searching the expressed sequence tag data base, we found a small sequence (Genbank TM accession no. AA103962) with similarity to the catalytic portion of mouse chondroitin-6-sulfotransferase, and obtained the corresponding cDNA fragment by reverse transcription-PCR (nucleotide numbers 1139 -1506 in Fig. 1A). Approximately 8 ϫ 10 5 plaques of a mouse day 7 embryo cDNA library were screened using the cDNA fragment as a probe, and six independent clones were obtained. The nucleotide sequence of the largest cDNA insert (2.2 kbp) was determined (Fig. 1A). The determined 2150-bp cDNA had a single open reading frame consisting of 483 amino acids, with a molecular mass of 52829 Da and four potential N-linked glycosylation sites (Fig.  1A). The sequence around the putative first ATG codon conformed to Kozak's rule (40), and the upstream region contained an in-frame stop codon. Hydropathy analysis indicated one prominent hydrophobic segment 20 residues in length in the amino-terminal region (Ala 8 -Leu 27 ), predicting that the protein has type II transmembrane topology (Fig. 1B). The predicted protein (mGlcNAc6ST) showed 25 and 27% sequence identity to mouse chondroitin-6-sulfotransferase (39) and human keratan sulfate Gal-6-sulfotransferase (10), respectively (Fig. 2). However, no significant homology in amino acid sequence was observed between the predicted protein and other known sulfotransferases (11)(12)(13)(14)(15).
Evidence That the Newly Cloned cDNA Encodes a Sulfotransferase-The expression plasmid derived from the cDNA, pcDNA3-GlcNAc6ST, was transfected into COS-7 cells, and the extracts of the transfected cells were assayed for sulfotransferase activity using 35 S-labeled PAPS as the sulfate donor and various glycoconjugates as sulfate acceptors. Chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, desulfated keratan sulfate, completely desulfated N-resulfated heparin, and mucin from porcine stomach and from bovine submaxillary gland did not serve as acceptors (data not shown). Since GlcNAc-6-O-sulfotransferases studied to date transfer sulfate only to GlcNAc residues exposed at the nonreducing end (22,23), we examined GlcNAc␤1-3Gal␤1-4GlcNAc as a sulfate acceptor. Superdex 30 chromatography of the reaction mixture indeed revealed a radioactive peak slightly larger than the acceptor, indicating that the acceptor was sulfated (Fig. 3A). The extract from untransfected cells or those transfected with the antisense cDNA showed much less activity (Table I). In contrast, Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc did not serve as an acceptor (Fig. 3B). This result may imply that sulfate was transferred to nonreducing GlcNAc. However, we could not deny the effect of the size difference of the oligosaccharides. Thus, we used a pentasaccharide, GlcNAc␤1-3Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc, as an acceptor and found that it was sulfated with the indistinguishable velocity as that for the trisaccharide. These results indicated that the cloned enzyme was a GlcNAc sulfotransferase.
The Sulfotransferase Transferred Sulfate to the 6 Position of the Nonreducing GlcNAc Residue-To determine the position to which 35 SO 4 was transferred to GlcNAc␤1-3Gal␤1-4GlcNAc, we cleaved the N-acetylglucosaminyl linkage of the radioactive product by N-deacetylation, deamination, and NaBH 4 reduction. On paper chromatography, the major radioactive product migrated at a position corresponding to sulfated 2,5-anhydromannitol (Fig. 4A), which is the expected product released from nonreducing sulfated GlcNAc. This result further established that the enzyme transferred sulfate to nonreducing GlcNAc. Then, we determined the position of sulfate in the sulfated 2,5-anhydromannitol. When the 35 S-labeled product was analyzed by HPLC, it was co-eluted with (SO 4 -6) 3 H-2,5-anhydromannitol (Fig. 4B). (SO 4 -3) 3 H-2,5-anhydromannitol was eluted at different fractions (Fig. 4B). The 35 S-labeled product also co-migrated with (SO 4 -6) 3 H-2,5-anhydromannitol on TLC (Fig. 4C). The 3-sulfo compound migrated faster than the 6-sulfo one under these conditions (Fig. 4C) (22). Although (SO 4 -4) 3 H-2,5-anhydromannitol was not available for comparison, it was described that the 3-sulfo and 4-sulfo compounds are indistinguishable owing to the symmetry of 2,5-anhydromannitol and have the same mobility upon TLC using the solvent system employed in this study (22). We also examined the structure of the 35 S-labeled sulfated 2,5-anhydromannitol by periodate oxidation. The 6-sulfo compound is expected to be cleaved, while the 3-sulfo as well as 4-sulfo compounds should be resistant. We observed that the 35 S-labeled sulfated 2,5anhydromannitol was cleaved after periodate oxidation, yielding a smear of band migrating slower than the sulfated 2,5anhydromannitol upon paper chromatography and that (SO 4 -3)2,5-anhydromannitol was resistant to the treatment (data not shown). Thus, the sulfated anhydromannitol was identified to be (SO 4 -6)2,5-anhydromannitol. In conclusion, 35 SO 4 was transferred to position 6 of GlcNAc residue located at the nonreducing end of GlcNAc␤1-3Gal␤1-4GlcNAc. Therefore, the cloned enzyme is a GlcNAc-6-O-sulfotransferase.
The GlcNAc-6-O-sulfotransferase Is Involved in the Synthesis of 6-Sulfo Sialyl N-Acetyllactosamine Antigen and 6-Sulfo Lewis X Antigen-We found that a significant portion of COS-7 cells transfected with the sense cDNA of GlcNAc-6-O-sulfotransferase expressed G72 antigen, the epitope of which is 6-sulfo sialyl N-acetyllactosamine (Fig. 5B). The parental cells (Fig. 5A) or cells transfected with the antisense cDNA (Fig. 5C) did not express the antigen. Neuraminidase digestion abolished the antigenicity (data not shown). Thus, we concluded that the sulfotransferase is involved in synthesis of 6-sulfo sialyl N-acetyllactosamine antigen.
As the next step, we doubly transfected COS-7 cells with the GlcNAc-6-O-sulfotransferase cDNA and fucosyltransferase IV cDNA. The latter enzyme is known to form Lewis X structure by transferring fucose to N-acetyllactosamine (41,42). The doubly transfected cells became positive for AG223 antigen, whose epitope is 6-sulfo Lewis X (Fig. 5F), while cells transfected with either cDNA alone did not (Fig. 5, D and E).
Northern Blot and Southern Blot Analyses-Among the adult mouse organs examined, GlcNAc-6-O-sulfotransferase was strongly expressed in the cerebrum, cerebellum, eye, lung, and pancreas (Fig. 6A). Moderate signals were also detected in the intestine, uterus, ovary, and peripheral as well as mesenteric lymph nodes (Fig. 6A). The size of the major transcript was 3.9 kb. On Southern blots, a single band was reactive with the sulfotransferase probe after digestion with EcoRI, EcoRV, or SacI, indicating that this GlcNAc-6-O-sulfotransferase was a single copy gene (Fig. 6B).
In Situ Hybridization Analysis-In situ hybridization was performed to determine the sites of the sulfotransferase expres-sion in mesenteric lymph nodes. HEV, where the ligand for L-selectin is located (43), are present in the paracortex (Fig. 7, A and B, arrows/arrowheads). They have a cuboidal endothelium with fairly large oval nuclei and a moderate amount of cytoplasm (Fig. 7D). The message of the sulfotransferase was specifically expressed in this endothelium (Fig. 7, B and E). DISCUSSION We cloned a cDNA encoding a GlcNAc-6-O-sulfotransferase. The enzyme transferred sulfate to an exposed GlcNAc residue ␤1-3 glycosidically linked to Gal␤1-4GlcNAc, but not to Glc-NAc residues contained in Gal␤1-4GlcNAc␤1-3Gal␤1-4GlcNAc. To date, GlcNAc-6-O-sulfotransferases have been studied using extracts or membrane preparations (22,23). The enzyme in extracts from human respiratory mucosa catalyzes sulfation of mucin carbohydrate chains (23), while that in the rat liver Golgi membrane is involved in the synthesis of NeuAc␣2-3(6)Gal␤1-4(SO 4 -6)GlcNAc, which is linked to the mannosyl core of asparagine-linked oligosaccharides (22). In both cases, GlcNAc residues at nonreducing ends serve as acceptors, but internal GlcNAc residues are poor acceptors. Thus, biosynthesis of the 6-sulfo N-acetyllactosamine structure has been concluded to start from 6-sulfation of GlcNAc followed by galactosylation by ␤1-4-galactosyltransferase (22,23). The acceptor specificity of the cloned sulfotransferase agrees with those of the sulfotransferases described above.
The cloned sulfotransferase was found to be involved in the synthesis of cell-surface antigenic epitopes, 6-sulfo sialyl Nacetyllactosamine and 6-sulfo Lewis X. These results indicated that the enzyme can be involved in the formation of 6-sulfo N-acetyllactosamine structures located in cell-surface glycoproteins and/or glycolipids. It remains to be clarified whether the enzyme also participates in biosynthesis of keratan sulfate.
Since 6-sulfo sialyl N-acetyllactosamine formed by the sulfotrasferase is converted to 6-sulfo sialyl Lewis X by fucosyltransferase VII, which is involved in expression of selectin ligands (44), our results have demonstrated that the cloned enzyme participates in formation of 6-sulfo sialyl Lewis X.
L-selectin located on lymphocytes plays a key role in their initial attachment to the HEV of lymph nodes (45). An Lselectin ligand in HEV of lymph nodes is GlyCAM-1, whose sulfation has been found to be required for its function (46). GlyCAM-1 has two sulfated N-acetyllactosamine structures, i.e. 6Ј-sulfo silayl Lewis X and 6-sulfo sialyl Lewis X, of which the former is the major structure (47). Whether 6Ј-sulfo sialyl Lewis X or 6-sulfo sialyl Lewis X is important for binding to L-selectin has not been settled at the present time (34,35,44,(47)(48)(49). However, the acceptor specificity of fucosyltransferase VII to sulfated sialyl N-acetyllactosamine (44) favors the view that 6-sulfo sialyl Lewis X is the endogenous ligand for Lselectin. Furthermore, anti-sialyl Lewis X antibodies that bind to 6-sulfo sialyl Lewis X reacted to HEV in lymph nodes and inhibited the binding of L-selectin to HEV, whereas antibodies that bind to 6Ј-sulfo sialyl Lewis X did not react to HEV (35). In view of the localization of GlcNAc-6-O-sulfotransferase transcripts in HEV of lymph nodes, the sulfotransferase is a likely candidate involved in sulfation of an L-selectin ligand.