©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Expression of Chick Chondrocyte Chondroitin 6-Sulfotransferase (*)

(Received for publication, March 20, 1995; and in revised form, May 15, 1995)

Masakazu Fukuta Kenji Uchimura Katsumi Nakashima Megumi Kato Koji Kimata (1) Tamayuki Shinomura (1) Osami Habuchi (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3`-phosphoadenosine 5`-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. The enzyme has been purified previously to apparent homogeneity from the serum-free culture medium of chick chondrocytes. The purified enzyme also catalyzed the sulfation of keratan sulfate. We have now cloned the cDNA of the enzyme. This cDNA contains a single open reading frame that predicts a protein composed of 458 amino acid residues. The protein predicts a Type II transmembrane topology similar to other glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases. Evidence that the predicted protein corresponds to the previously purified C6ST was the following: (a) the predicted sequence of the protein contains all of the known amino acid sequence, (b) when the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, both the C6ST activity and the keratan sulfate sulfotransferase activity were overexpressed, (c) a polyclonal antibody raised against a fusion peptide, which was expressed from a cDNA containing the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST, and (d) the predicted protein contained six potential sites for N-glycosylation, which corresponds to the observation that the purified C6ST is an N-linked glycoprotein. The amino-terminal amino acid sequence of the purified protein was found in the transmembrane domain, suggesting that the purified protein might be released from the chondrocytes after proteolytic cleavage in the transmembrane domain.


INTRODUCTION

Chondroitin sulfate proteoglycan is abundantly found in cartilage and is thought to contribute to the expression and the maintenance of the phenotype of chondrocytes(1) . Chondroitin sulfate proteoglycan is also present in the various tissue other than cartilage and thought to play an important role in cellular interaction(2) . Major chondroitin sulfate found in the mammalian or avian tissues bears sulfate groups at position 6 or position 4 of acetylgalactosamine residue. The ratio of 6-sulfation/4-sulfation varies with development of animals(3, 4) , malignant change(5) , or susceptibility to atherosclerosis(6) . The presence of two sulfate groups per one repeating disaccharide unit was also reported. The GalNAc(4,6-bis-SO(4)) residue was found in chondroitin sulfate from mast cell(7) , rat glomeruli(8) , and subcultured chick chondrocytes(9) . GalNAc(4,6-bis-SO(4)) located at the nonreducing end was found in chondroitin sulfate from rat cartilage(10) , cultured chick chondrocytes(11) , and thrombomodulin(12) . GlcA(2SO(4))-GalNAc(6SO(4)) unit was contained in chondroitin sulfate from cultured mast cells (13) . Such observed diversity in sulfation patterns of chondroitin sulfate may reflect the molecular basis of its function. As reported in heparan sulfate N-sulfotransferase(14, 15) , chondroitin 6-sulfotransferase(16) , and heparan sulfate 6-sulfotransferase(17) , a specific sulfotransferase seems to be involved in the sulfation of the particular position of sugar residues of glycosaminoglycans. On the other hand, Wlad et al.(18) presented evidence suggesting that the same enzyme may catalyze the sulfation of both 2-O of L-iduronic acid residue and 6-O of D-glucosamine residue of heparin. Cloning and expression of glycosaminoglycan sulfotransferase seems to offer the direct evidence about the acceptor substrate specificity. Of various glycosaminoglycan sulfotransferases, N-sulfotransferase/N-deacetylase has been cloned from rat liver(19) , heparin-producing cell line(20) , and mouse mastocytoma (21) and expressed in COS cells.

We have previously purified chondroitin 6-sulfotransferase (C6ST), (^1)which catalyzes the transfer of sulfate from 3`-phosphoadenosine 5`-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin, to apparent homogeneity(16) . This enzyme was also found to catalyze the sulfation of keratan sulfate. In this paper we report the cloning of the cDNA encoding chick chondrocyte C6ST and the expression of it in COS-7 cells.


EXPERIMENTAL PROCEDURES

Assay and Purification of Chondroitin 6-Sulfotransferase

C6ST was purified from the serum-free culture medium of chick embryo chondrocytes as described previously(16) . Activity of C6ST, C4ST, and KSST was determined as described previously (16) .

Digestion of the Purified Sulfotransferase with N-Glycanase

The purified sulfotransferase was precipitated with 10% trichloroacetic acid. The precipitates were washed with acetone and digested with recombinant N-glycanase (Genzyme) by the methods recommended by the manufacturer. After digestion, the reaction mixture was analyzed by SDS-PAGE as described by Laemmli(22) .

Determination of Amino Acid Sequence of the Purified Protein

The purified C6ST (10 µg as protein) was digested with N-glycanase as described above for 12 h. The resulting deglycosylated C6ST and the intact C6ST (20 µg as protein) were subjected to 10% SDS-PAGE and transferred to a PVDF membrane. After the membrane was stained with Coomassie Blue, the bands of the 49- and 47-kDa protein formed after N-glycanase digestion and the band of the 75-kDa intact C6ST were cut out and used for amino acid sequencing. The limited digestion of the purified C6ST with protease V8 was carried out according to Cleveland et al.(23) . Briefly, the purified protein (30 µg) was separated with SDS-PAGE using 10% gel. After staining the gel with Coomassie Blue, the 75-kDa protein band was cut out and inserted in the wells of another 16% gel. After a buffer containing protease V8 (sequencing grade, Boehringer Mannheim) in the ratio of 0.05 µg/µg of the purified protein was layered on the inserted gel, SDS-PAGE was started. When the dye front reached the edge of the separation gel, the power was turned off. After 30 min, electrophoresis was resumed. The peptides formed by the limited protease digestion were transferred to a PVDF membrane. After staining the membrane with Coomassie Blue, the 19-kDa peptide band was excised. The PVDF membrane containing the protein and peptide samples were sent to Takara Shuzo Co. Ltd., Kyoto, Japan for determining the amino-terminal amino acid sequence.

Oligonucleotides and Polymerase Chain Reaction

Degenerated oligonucleotide primers were designed as indicated in Fig. 2A. Two sense primers (1s and 2s) and one antisense primer (3a) were prepared from the amino acid sequence of the 75-kDa protein and the 19-kDa peptide, respectively. At 5`-end of oligonucleotide 1s and 3a, restriction enzyme recognition sites were introduced; HindIII site for 1s and EcoRI site for 3a. The first strand of cDNA was synthesized by the reverse transcriptase reaction using poly(A) RNA from chondrocyte as a template and degenerated oligonucleotide 3a as a primer. The PCR reaction was carried out in a final volume of 100 µl containing 50 pmol each of oligonucleotide 1s and 3a, 10 µl of the reverse transcriptase reaction mixture in which the first strand cDNA was synthesized, 100 µM each of four deoxynucleoside triphosphates, 2.5 units of AmpliTaq polymerase (Perkin-Elmer). Amplification was carried out by 30 cycles of 94 °C for 1 min, 45 °C for 1 min, and 55 °C for 3 min.


Figure 2: A, oligonucleotide primer sequences, derived from peptide 1 and 2, used for the PCR experiment shown in Fig. 2B. At the 5`-end of oligonucleotide 1s and 3a, recognition site of HindIII and EcoRI, respectively, were introduced. B, agarose gel electrophoresis of the PCR products. Lane 1, the template was poly(A) RNA from chondrocytes; lane 2 and 3, the template was the 400-bp PCR product of lane 1 which was indicated by an arrowhead.



Reaction products were subjected to agarose gel electrophoresis (Fig. 2B). The amplified DNA band (indicated by an arrowhead in Fig. 2B) was cut out and the DNA fragment was recovered from the gel, digested with HindIII and EcoRI, and subcloned into these sites of Bluescript (Stratagene). Subclones were characterized by sequencing.

The radioactive probe for screening the cDNA library was prepared from the PCR product by the random oligonucleotide-primed labeling method (24) using [alpha-P]dCTP (Amersham Corp.) and a DNA random labeling kit (Takara Shuzo).

Construction of gt11 Library

Total RNA was prepared from the chick embryo chondrocytes cultured for 11 days in DMEM containing 10% fetal bovine serum as described previously (25, 26, 27) by the guanidine thiocyanate/CsCl methods(28) . Poly(A) RNA was purified by oligo(dT)-cellulose column chromatography. The synthesis of cDNA and ligation of the cDNA to EcoRI-digested gt11 (Pharmacia) was carried out using TimeSaver cDNA synthesis kit (Pharmacia). Random oligonucleotide primers were used for the reverse transcriptase reaction. The ligated DNA was packaged in vitro using a Stratagene Gigapack II packaging extract and plated on Escherichia coli Y1088. The library was used for cDNA screening without further amplification.

Screening of gt11 Library

Approximately 5 10^5 plaques were screened. Hybond N nylon membrane (Amersham) replicas of the plaques from the gt11 cDNA library were fixed by the alkali fixation method recommended by the manufacturer, prehybridized in a solution containing 50% formamide, 5 SSPE (SSPE, sodium chloride/sodium phosphate/EDTA buffer), 5 Denhardt's solution, 0.5% SDS, 0.04 mg/ml of denatured salmon sperm DNA, and 0.004 mg/ml E. coli DNA for 3.5 h at 42 °C. Hybridization was carried out in the same buffer containing P-labeled probe for 16 h at 42 °C. The filters were washed at 55 °C in 1 SSPE, 0.1% SDS, and subsequently in 0.1 SSPE, 0.1% SDS, and positive clones were detected by autoradiography.

DNA Sequence Analysis

DNA from gt11 positive clones were isolated and cut with EcoRI, which excised the cDNA insert in a single fragment. The fragments were inserted into Bluescript plasmid, and deletion clones were prepared as described (29, 30) using a DNA deletion kit (Takara Shuzo). The complete nucleotide sequence was determined independently on both strands using the dideoxy chain termination method (31) with [alpha-P]dCTP and Sequenase (U. S. Biochemical Corp.). DNA sequences were compiled and analyzed using the Gene Works computer programs (IntelliGenetics).

Construction of pCXNC6ST

To construct the plasmid containing the C6ST cDNA named pCXNC6ST, the EcoRI fragment containing the 2354-bp cDNA indicated in Fig. 3A was excised from the Bluescript plasmid and ligated into the EcoRI site of pCXN2 expression vector (pCXN2 vector was constructed by Dr. Jun-ichi Miyazaki, Department of Disease-related Gene Regulation, Faculty of Medicine, University of Tokyo (32) and was a gift from Dr. Yasuhiro Hashimoto, Tokyo Metropolitan Institute of Medical Sciences). JM109 cells were transformed with the ligation mixture and plated on LB ampicillin plates. Recombinant plasmids were analyzed by restriction mapping using BamHI to confirm the correct orientation of pCXNC6ST. The recombinant plasmids used for the transfection were purified with CsCl/ethidium bromide equilibrium centrifugation three times. The plasmid that contained the cDNA fragment in the reversed orientation was named as pCXNC6ST2 and used for control experiments.


Figure 3: Nucleotide sequence of the C6ST cDNA, and the predicted amino acid sequence and hydropathy plot of the protein. A, the predicted amino acid sequence is shown below the nucleotide sequence. Peptides from which amino acid sequence data were obtained are underlined. Six potential N-linked glycosylation sites are underlined with double solid lines. The putative transmembrane hydrophobic domain is boxed. The site at which C6ST might be cleaved during the secretion is indicated with a triangle. B, the hydropathy plot was calculated by the method of Kyte and Doolittle (34) with a window of 11 amino acids.



Transient Expression of Chondroitin 6-Sulfotransferase cDNA in COS-7 Cells

COS-7 cells (obtained from Riken Cell Bank, Tsukuba, Japan) were plated in 100-mm culture dishes at a density of 8 10^5 cells/dish. The volume of the medium was 10 ml. The medium used was DMEM containing penicillin (100 units/ml), streptomycin (50 µg/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 10^6 cells/dish (48 h after plating), COS-7 cells were transfected with pCXNC6ST or pCXNC6ST2. The transfection was performed using the DEAE-dextran method(33) . 5 ml of the prewarmed DMEM containing 10% Nu serum (Collaborative Biomedical Products) was mixed with 0.2 ml of PBS containing 10 mg/ml DEAE-dextran plus 2.5 mM chloroquine solution. 15 µg 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 PBS. After the cells were left at room temperature for 2 min, the dimethyl sulfoxide solution was aspirated, and 25 ml of DMEM containing penicillin (100 units/ml), streptomycin (50 µg/ml), and 10% fetal bovine serum was added. The cells were incubated for 67 h, washed with DMEM alone, scraped, and homogenized with a Dounce homogenizer in 1.5 ml/dish of 0.25 M sucrose, 10 mM Tris-HCl, pH 7.2, and 0.5% Triton X-100. The homogenates were centrifuged at 10,000 g for 20 min, and the activities of C6ST, C4ST, and KSST in the supernatant fractions were measured.

Preparation of Polyclonal Antibody against a Fusion Peptide

A DNA fragment which codes for 150 amino acid residues (Glu to Ile shown in Fig. 3A) was amplified by PCR using the 465-bp DNA fragment as a template which was amplified from poly(A) RNA as described above. At 5`-end of the oligonucleotide primers, restriction enzyme recognition sites were introduced: BamHI site for the sense primer beginning at Glu and EcoRI site for the antisense primer beginning at Ile. Amplification was carried out by 30 cycles of 94 °C for 1 min, 45 °C for 2 min, and 72 °C for 2 min. The PCR product was digested with EcoRI and BamHI and subcloned into these sites of pRSET A plasmid (Invitrogen). The resulting plasmid was transfected in E. coli DE3, and the fusion peptide produced was purified by a ProBond Resin (Invitrogen) by the method described by the manufacturer. The fusion peptide purified with the affinity column was still contaminated by other proteins. The final purification of the fusion peptide was achieved with 15% SDS-PAGE. The 22-kDa fusion peptide eluted from the polyacrylamide gel with 50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 100 mM EDTA, 0.1% SDS, and 5 mM dithiothreitol was precipitated with 5 volumes of acetone. The precipitate was dissolved in 50 mM Tris-HCl, pH 8.0, containing 6 M guanidine HCl, 150 mM NaCl, 0.1 mM EDTA, 0.1% Nonidet P-40, and 1 mM dithiothreitol and dialyzed against PBS. The dialyzed sample was injected into mice intraperitoneally. Polyclonal antibody was obtained after twice boost injections.

Immunoblotting

The purified C6ST and the 22-kDa fusion peptide were subjected to 12% SDS-PAGE and transferred to a nitrocellulose filter. Protein bands were visualized by Amido Black. For immunostaining, the filter was blocked and incubated with the diluted antiserum (1:1000) raised against the 22-kDa fusion peptide as described above. Bound antibodies were visualized with an enzyme reaction using peroxidase-conjugated anti-mouse immunoglobulin goat IgG (Cappel) as a secondary antibody.

Immunoprecipitation of C6ST

The mouse antiserum (3 µl) against the 22-kDa fusion peptide was added to 31 µl of Buffer A (10 mM Tris-HCl, pH 7.2, 130 mM NaCl, 10 mM MgCl(2), 2 mM CaCl(2), 0.1% Triton X-100, 20% glycerol) containing 23 ng of the purified C6ST. The mixtures were incubated at 4 °C overnight. 6 µl of anti-mouse IgG rabbit IgG (Cappel) was added to the mixtures. After further incubation for 1 h at 0 °C, the reaction mixtures were mixed with 20 µl of a 50% (v/v) suspension of protein A-Sepharose (Pharmacia), which was equilibrated with Buffer A, and were rocked for 30 min at 4 °C. The immune complexes on the protein A beads were removed by centrifugation and C6ST activity remaining in the supernatant solution was assayed.

Northern Blot Hybridization

Poly(A) RNA prepared from chick chondrocytes as described above was denatured in 50% formamide (v/v), 5% formaldehyde (v/v), 20 mM MOPS, pH 7.0, at 65 °C for 10 min, electrophoresed in 1.2% agarose gel containing 5% formaldehyde (v/v), and transferred to a Hybond N nylon membrane overnight. The RNA was fixed by baking at 80 °C for 2 h and then prehybridized in a solution containing 50% formamide, 5 SSPE, 5 Denhardt's solution, 0.5% SDS, and 0.1 mg/ml of denatured salmon sperm DNA for 3 h at 42 °C. Hybridization was carried out in the same buffer containing P-labeled probe for 14 h at 42 °C. The radioactive probe was the same as the probe used for the screening of the cDNA library described above. The filters were washed at 65 °C in 2 SSPE, 0.1% SDS, and subsequently in 1 SSPE, 0.1% SDS. The membrane was exposed to x-ray film for 26 h with a intensifying screen at -80 °C.


RESULTS

Amino Acid Sequence of Chondroitin 6-Sulfotransferase

The purified C6ST gave a broad protein band on SDS-PAGE (Fig. 1, lane 1). Microheterogeneity of N-linked oligosaccharides attached to the enzyme probably cause the width of the band. After the sulfotransferase was digested with N-glycanase, the protein band of 75 kDa disappeared, and two sharp bands (49 and 47 kDa) appeared as shown in Fig. 1, lane 2-4. A broad protein band of 56 kDa appeared to be a partially N-deglycosylated product, since the band became weaker as the period of the digestion with N-glycanase became longer. The amino-terminal amino acid sequence of the 49-kDa protein was identical with that of the 47-kDa protein as far as the identification was possible; therefore we determined the amino-terminal amino acid sequence of the 75-kDa intact C6ST without further separation (Table 1). We also prepared the 19-kDa peptide from the intact protein by the limited digestion with protease V8, and the amino acid sequence of the 19-kDa peptide was determined (Table 1).


Figure 1: N-Glycanase digestion of the purified chondroitin 6-sulfotransferase. Chondroitin 6-sulfotransferase (0.6 µg as protein) was digested with N-glycanase as described under ``Experimental Procedures,'' and 0.2 µg of protein was analyzed by SDS-PAGE. Proteins were visualized with silver nitrate stain. Lane 1, undigested control; lane 2, protein digested with N-glycanase for 1 h; lane 3, protein digested with N-glycanase for 2 h; lane 4, protein digested with N-glycanase for 12 h. Molecular size standards were the following: myosin (205 kDa), beta-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa). 2-Mercaptoethanol was removed from sample buffer for SDS-PAGE to avoid artifact of silver staining. The arrowhead indicates the position of N-glycanase.





Generation of PCR Probe to Screen cDNA Library

When primer 1s and 3a (for the nucleotide sequences of the primers, see Fig. 2A) were used in a PCR with poly(A) RNA from chick chondrocytes as template, DNA fragments of 400 and 600 bp were obtained (Fig. 2B, lane 1). The 400-bp product, which is indicated by an arrowhead in Fig. 2B, lane 1, appeared to be specific because, when primer 2s and 3a were used in a PCR with the 400-bp fragment as template, a fragment slightly shorter than the template was amplified (Fig. 2B, lane 2). Further proof that the 400-bp fragment was amplified from the mRNA of the purified C6ST protein was obtained by sequencing the 400-bp fragment; the fragment contained 465 nucleotides in which a nucleotide sequence corresponding to the amino acid sequence encoded by primer 2s was present adjacent to the sequence corresponding to primer 1s.

Screening of gt11 Library

The above described 465-bp PCR product was labeled with [alpha-P]dCTP by the random oligonucleotide-primed labeling method and used as a probe to screen a gt11 library obtained as described under ``Experimental Procedures.'' About 90 positive clones were observed from 5 10^5 plaques. Sixteen independent clones were isolated and subcloned into Bluescript. The nucleotide sequence of the largest cDNA insert (2.3 kilobases) was determined.

cDNA and Predicted Protein Sequence of the Chondroitin 6-Sulfotransferase

The nucleotide sequence of the C6ST cDNA and the predicted amino acid sequence are shown in Fig. 3A. The amino-terminal sequence contains two in-frame ATG codons. A single open reading frame beginning at the first ATG codon predicts a protein of 458 amino acid residues with a molecular mass of 52,193 Da with six potential N-linked glycosylation sites. 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 amino-terminal region, 14 residues in length, that extends from amino acid residues 24-37 (Fig. 3B). The amino-terminal-amino acid sequence of the purified C6ST was found in the transmembrane domain. The molecular mass of the truncated protein at the site in the transmembrane domain was calculated as 47,885 Da, and it agreed well with the molecular mass of the protein formed after N-glycanase digestion. All the amino acid sequences that have been obtained from the purified protein were found in the predicted protein sequences, confirming that the cDNA clone encodes the purified C6ST protein.

Expression of Chondroitin 6-Sulfotransferase cDNA in COS-7 Cells

Direct evidence demonstrating that the isolated cDNA encodes the chondroitin 6-sulfotransferase protein was obtained by expressing it in COS-7 cells. COS-7 cells were transfected with the pCXNC6ST, a recombinant plasmid containing the isolated cDNA in the mammalian expression vector pCXN2. The transfected cells were scraped at 67 h after transfection, homogenized, and centrifuged. Activities of C6ST, C4ST, and KSST contained in the supernatant fractions were determined in the presence or absence of sulfate acceptors. Control experiments without vector, and with vector containing the cDNA in the reversed orientation (pCXNC6ST2), were also done. As shown in Table 2, when the vector containing the isolated cDNA was used, C6ST activity and KSST activity in the transfected cells was 20- and 9-fold, respectively, above that of the control transfections. In contrast, C4ST activity in the transfected cells was not increased at all. These results demonstrate that the isolated cDNA encodes a protein with both the C6ST activity and the KSST activity.



Immunoblotting and Immunoprecipitation

A polyclonal antibody raised against the 22-kDa fusion peptide, which was expressed from the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST with molecular mass of 75 kDa (Fig. 4, lane 2). This antibody did not stain any proteins contained in the culture medium of chondrocytes (lane 1). C6ST, which was contained in the culture medium, was not detected on the immunoblot; this result may be due to the trace amount of C6ST in the culture medium. When the antibody was added to the purified C6ST solution and the immunocomplexes on protein A were removed by centrifugation, a large part of the C6ST activity was depleted from the soluble fractions (Table 3). These results clearly indicate that C6ST contains a polypeptide sequence whose antigenicity is indistinguishable from the 22-kDa fusion peptide and confirm that the isolated cDNA encodes C6ST protein.


Figure 4: Immunoblots of the purified C6ST and the 22-kDa fusion peptide. Lanes 1 and 4, 12 µg (as protein) each of the serum-free culture medium of chondrocytes; lanes 2 and 5, 0.4 and 0.8 µg, respectively, of the purified C6ST; and lanes 3 and 6, 0.1 and 0.5 µg, respectively, of the 22-kDa fusion peptide. Lanes 1-3, immunoblot stained with anti-22-kDa fusion peptide polyclonal antibody; lanes 4-6, Amido Black staining. Molecular size standards were the following: bovine serum albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20.1 kDa), and alpha-lactalbumin (14.2 kDa).





Northern Analysis

A Northern blot of poly(A) RNA from the cultured chick chondrocytes was made and hybridized with a radioactive probe prepared from the PCR-amplified DNA fragment with 465 bp described above by the random oligonucleotide-primed labeling method. As can be seen in Fig. 5, four bands with a relative size of 5.8, 4.5, 3.2, and 2.5 kilobases were obtained.


Figure 5: Northern blot analysis. Polyadenylated RNA (5 µg) prepared from the cultured chick embryo chondrocytes was subjected to Northern blot analysis, using hybridization and wash conditions described under ``Experimental Procedures.'' The blot was probed with the 465-bp fragment that was obtained by PCR. The arrowheads indicate the position of different mRNAs: 5.8, 4.5, 3.2, and 2.5 kilobases. The positions of ribosomal RNAs are indicated at the left.




DISCUSSION

We have cloned a cDNA that encodes the C6ST. Different lines of evidence indicated that the cloned cDNA corresponds to the C6ST previously purified from the culture medium of chondrocytes: (a) the predicted sequence of the protein contains all of the known amino acid sequence, (b) both the C6ST activity and the KSST activity were overexpressed when the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, (c) a polyclonal antibody raised against a fusion peptide, which was expressed from a cDNA containing the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST, and (d) the predicted protein contained six potential N-linked glycosylation sites, which fits with the observation that the purified C6ST is an N-linked glycoprotein. The predicted protein showed no sequence homology to other sulfotransferases, including N-sulfotransferase/N-deacetylases. The sequence observed in most of sulfotransferases, GXXGXXK(R) (19) , was not found in C6ST. LEKCGR, which was reported as a peptide sequence at the biding site for PAPS in aryl sulfotransferase IV(35) , was not present in C6ST.

The amino-terminal sequence contains two in-frame ATG codons. When the sequence surrounding the first ATG codon is compared to the eukaryotic consensus translation sequence(36) , the purine at -3, is not conserved, but G in position +4 is conserved. Kozak showed that, in the absence of a purine in position -3, G in position +4 was essential for efficient translation(37) . The sequence surrounding the second ATG codon (Met in Fig. 3A) also partially fits with the consensus sequence; position -3 of the second ATG codon is A, whereas position +4 is not G but A. It remains to be studied whether both of the two in-frame ATG codon contained in C6ST cDNA could function as the initiation codon.

The hydrophobic transmembrane domain of C6ST contains 14 amino acid residues; the length of the transmembrane domain of C6ST seems to be shorter than those of most of cloned glycosyltransferases(38) . Eckhardt et al.(39) , however, recently reported that a stretch of 13 hydrophobic amino acids was found within the NH(2)-terminal domain of eukaryotic polysialyltransferase-1, and they supposed that the hydrophobic domain represents a Golgi retention signal, because the cationic borders characteristic for type II transmembrane proteins were also found. Cationic amino acid residues (Lys and Lys in Fig. 3A) were also found at both sides of the hydrophobic domain of C6ST. Since the microsomal C6ST from the cultured chondrocytes was markedly activated by detergents such as Triton X-100 or Brij 58(40, 41, 42) , C6ST appeared to be a membrane protein, although the transmembrane domain seemed to be rather unusual. The amino-terminal amino acid sequence of the purified C6ST was found in the predicted transmembrane domain, suggesting that the purified protein might be released from the chondrocytes after proteolytic cleavage within the transmembrane domain.

When COS-7 cells were transfected with the expression vector containing the cDNA of C6ST, C6ST activity in the transfected cells was 20-fold above that of the control transfections, but C4ST activity in the cells was not increased at all, indicating that C4ST is quite a different enzyme from C6ST. We found previously that the purified C6ST catalyzed the transfer of sulfate to keratan sulfate (16) and that position 6 of the Gal residue of keratan sulfate was sulfated by the purified C6ST. (^2)The sulfotransferase activity toward keratan sulfate was also expressed when pCXNC6ST was transfected to COS-7 cells, suggesting that position 6 of GalNAc residue in chondroitin and position 6 of Gal residue in keratan sulfate may be sulfated by the same enzyme.


FOOTNOTES

*
This work was supported by the Grant-in-aid for Scientific Research on Priority Areas No. 06267210 from the Ministry of Education, Science, and Culture, Japan and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) D49915[GenBank].

§
To whom correspondence should be addressed: Dept. of Life Science, Aichi University of Education, Kariya 448, Japan. Fax.: 81-566-36-4337.

^1
The abbreviations used are: C6ST, chondroitin 6-sulfotransferase; C4ST, chondroitin 4-sulfotransferase; KSST, keratan sulfate sulfotransferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PAPS, 3`-phosphoadenosine 5`-phosphosulfate; MOPS, 4-morpholinopropanesulfonic acid; PBS, phosphate-buffered saline.

^2
O. Habuchi and Y. Hirahara, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Jun-ichi Miyazaki, Department of Disease-related Gene Regulation, Faculty of Medicine, University of Tokyo and Dr. Yasuhiro Hashimoto, Tokyo Metropolitan Institute of Medical Sciences, for donating pCXN2 expression vector; Dr. Keiichi Yoshida, Tokyo Research Institute of Seikagaku Corp., for generous gift of bovine corneal keratan sulfate; and Dr. Naomi Yamakawa, Institute for Molecular Medical Science, Aichi Medical University, for useful suggestions about the pRSET vector.


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