JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M002443200 on April 25, 2000

J. Biol. Chem., Vol. 275, Issue 26, 20188-20196, June 30, 2000
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Molecular Cloning and Expression of Two Distinct Human Chondroitin 4-O-Sulfotransferases That Belong to the HNK-1 Sulfotransferase Gene Family*

Nobuyoshi Hiraoka, Hiroaki NakagawaDagger, Edgar Ong, Tomoya O. Akama, Michiko N. Fukuda, and Minoru Fukuda§

From the Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037

Received for publication, March 23, 2000, and in revised form, April 24, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using an expression cloning strategy, the cDNA encoding the human HNK-1 sulfotransferase (HNK-1ST) has been cloned. During this cloning we found that HNK-1ST and other Golgi-associated sulfotransferases cloned before share homologous sequences including the RDP motif (Ong, E., Yeh, J.-C., Ding, Y., Hindsgaul, O., and Fukuda, M. (1998) J. Biol. Chem. 223, 5190-5195). Using this conserved sequence in HNK-1ST as a probe, we identified two expressed sequence tags in EST data base which have 31.6 and 30.7% identity with HNK-1ST at the amino acid levels. Expression of these two full-length cDNAs failed to form HNK-1 glycan nor to add sulfate to CD34 or NCAM. Surprisingly, proteins expressed by these cDNAs transferred sulfate to the C-4 position of N-acetylgalactosamine in chondroitin and desulfated dermatan sulfate, thus we named these two enzymes, chondroitin 4-O-sulfotransferase 1 and -2 (C4ST-1 and C4ST-2). Both C4ST-1 and C4ST-2, however, did not form 4,6-di-O-sulfated N-acetylgalactosamine when chondroitin sulfate C was used as an acceptor. Moreover, analysis of 35S-labeled dermatan sulfate formed by C4ST-1 indicate that sulfation preferentially took place in GlcAright-arrowGalNAc unit than in IdoAright-arrowGalNAc unit, suggesting that 4-O-sulfation at N-acetylgalactosamine may precede epimerization of glucuronic acid to iduronic acid during dermatan sulfate biosynthesis. Northern analysis demonstrated that the transcript for C4ST-1 is predominantly expressed in peripheral leukocytes and hematopoietic tissues while the C4ST-2 transcript is more widely expressed in various tissues. These results indicate C4ST-1 and C4ST-2 play complementary roles in chondroitin and dermatan sulfate synthesis in different tissues.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sulfate groups in carbohydrates play important roles in conferring highly specific functions on glycoproteins, glycolipids, and proteoglycans (1-3). Expression of certain sulfated carbohydrates is spatially and temporally controlled, thereby providing developmental regulation of those functions displayed by such sulfation. One of these sulfated glycans is the HNK-1 glycan (4, 5). The functional significance of HNK-1 glycan was first recognized as an antigen involved in peripheral demyelinative neuropathy. The structural analysis of glycolipids reactive with the autoantibodies led to the discovery that the HNK-1 epitope is sulforight-arrow3GlcAbeta 11right-arrow3Galbeta 1right-arrow4GlcNAcbeta right-arrowR (6, 7).

Subsequently, HNK-1 glycan has been found in a number of neural cell adhesion molecules, including NCAM, myelin-associated glycoprotein, L1, contactin, and Po (5, 8-11). Using monoclonal antibodies or isolated carbohydrates, various laboratories reported that HNK-1 glycan is involved in cell-cell and cell-substratum interactions (12, 13). In one study, a non-sulfated form of HNK-1 precursor glycan did not facilitate neurite outgrowth as opposed to a functional, intact HNK-1 glycan (13). These results, combined together, suggest that HNK-1 glycan plays critical roles in development, in particular during neural cell development.

The HNK-1 carbohydrate is synthesized by the addition of a sulfate to beta 1,3-glucuronylated N-acetyllactosamine, GlcAbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcright-arrowR (4). Recently, we and others cloned the cDNA encoding HNK-1 sulfotransferase using an expression cloning strategy (14, 15). During this cloning, we discovered that the newly cloned HNK-1 sulfotransferase and other Golgi-associated sulfotransferases cloned before share a common sequence motif, which includes ZZRDPXXXZ, where X and Z denote any amino acid and hydrophobic amino acids, respectively (14). Subsequently, it was revealed that this sequence motif is a part of the binding site for 3'-phosphate group of the donor substrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) (16, 17). Most recent studies showed that the arginine residue (Arg) in the RDP motif is involved in hydrogen bonding to 3'-phosphate group while aspartic acid (Asp) and proline (Pro) residues participate in the core structure of the 3'-phosphate-binding site by residing in a tight turn of the polypeptides (17, 18). In addition, the amino acid sequences responsible for binding to 5'-phosphosulfate are conserved among different sulfotransferases (17).

The presence of the above weak but discernible similarity among different sulfotransferases suggested a possibility that other sulfotransferases may be identified by their similarity to sulfotransferases cloned already. In fact, we and others cloned the cDNA encoding L-selectin ligand sulfotransferases that add a sulfate to the 6-position of N-acetylglucosamine, which is eventually converted to 6-sulfo sialyl Lewis X, NeuNAcalpha 2right-arrow3Galbeta 1right-arrow4[sulforight-arrow6(Fucalpha 1right-arrow3)GlcNAc]beta 1right-arrow6R (19-21). This cloning was achieved by searching the EST data base for cDNAs related to chondroitin sulfate 6-O-sulfotransferase (22) and keratan sulfate Gal-6-O-sulfotransferase (23).

In the present study, we first describe the isolation of two isoforms of cDNAs by screening the EST data base for cDNAs related to the human HNK-1 sulfotransferase (14). The expression of full-length cDNAs unexpectedly revealed that these cDNAs encode novel chondroitin 4-O-sulfotransferases, adding a sulfate to 4-position of N-acetylgalactosamine residues in chondroitin and desulfated dermatan sulfate. Moreover, we found that these two chondroitin 4-sulfotransferases exhibit diverse tissue distribution, indicating that these two enzymes play complementary roles in different tissues.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of cDNAs Encoding Chondroitin 4-O-Sulfotransferases-- In HNK-1ST, the conserved motif, IVRDPFERL residues in amino acid residues 187-195 (14). The amino acid sequence of residues 165-230, which includes the above motif, was thus used as a probe to search dbEST using the TBLASTN program. Initially, two query genes AA310375 and AA233362 were identified, which had 50% in 50 amino acids and 69% in 26 amino acids identity with HNK-1ST, respectively.

After blast search for a sequence homologous to AA310375, AA744877 was identified. AA744877 is a cDNA prepared from germinal center B lymphocytes. Sequence analysis of this cDNA, obtained from Genome Systems (St.Louis, MS), revealed that this cDNA encodes a protein consisting of 352 amino acids. The cDNA also contains 5'-untranslated sequence (150 base pairs) and 3'-untranslated sequence (330 base pairs). The cDNA insert was digested with HindIII and XhoI and cloned into the same sites of pcDNA3.1/Hygro (Invitrogen, Carlsbad, CA), resulting in pcDNA3.1-C4ST-1 (the name of C4ST-1 was given after the determination of acceptor specificity).

The second gene was initially identified in AA233362 and AA777237 derived from the human NT2 cell line and SS20w fetal liver/spleen. Since these two clones lacked the 5'-region, 5'-rapid amplification of cDNA ends was carried out using poly(A)+ RNA from human lymph nodes (CLONTECH, Palo Alto, CA). However, a new EST sequence, AA182585, which contained the full coding sequence was released in the meantime. The cDNA was thus excised from AA182585 with BamHI and XhoI and cloned into the same sites of pcDNA3.1/Hygro, resulting in pcDNA3.1-C4ST-2.

pcDNAI-HNK1ST harboring the cDNA encoding a human HNK-1 sulfotransferase was cloned as described previously (14). The cDNA encoding chondroitin 6-O-sulfotransferase (C6ST) was cloned by reverse transcriptase-PCR using poly(A)+ RNA isolated from mouse embryo (E17), as described previously (20). The 5'- and 3'-primers used in this PCR correspond to nucleotides -67 to -49 (1-3 encode for the initiation methionine) and nucleotides 1518-1527, respectively (24), and also contained XhoI and HindIII sites, respectively. The PCR products were cloned into pBluescript by TA cloning. The resultant cDNA was excised by XbaI and HindIII and cloned into the same sites of pcDNA3.1/Zeo, resulting in pcDNA3.1-C6ST. pcDNA3-GlcAT-P encoding beta 1,3-glucuronyltransferase that forms the HNK-1 precursor glycan (25) was cloned as described before (14).

Sulfotransferase Assay-- CHO cells were transfected with pcDNA3.1-C4ST-1, pcDNA3.1-C4ST-2, pcDNA3.1-C6ST, or pcDNAI -HNK-1ST using LipofectAMINE PLUS (Life Technologies, Inc., Rockville, MD). Sixty-two h after transfection, the cells attached to plates were washed with phosphate-buffered saline, scraped, and homogenized in 10 mM Tris-HCl, pH 7.2, containing 0.5% Triton X-100, 0.25 M sucrose, a protease inhibitor mixture, and 1 mM aprotinin as described previously (22). The homogenate was mixed well by rotation for 1 h, then centrifuged at 10,000 × g for 15 min. The supernatant derived from the transfected CHO cells and mock-transfected CHO cells were used as the enzyme source.

Chondroitin sulfate 4- and 6-O-sulfotransferase and heparan sulfate sulfotransferase activities were assayed as described previously (26). Briefly, the reaction mixture (50 µl) contained 50 mM imidazole-HCl, pH 6.8, 0.005% protamine chloride, 2 mM dithiothreitol, 50 µg of acceptor glycosaminoglycans, 2 µM [35S]PAPS (about 5 × 105 cpm), and 25 µl of an enzyme solution. After incubation for 1 h at 37 °C, the reaction mixture was boiled for 2 min, then 0.1 volume of 4 M potassium acetate and 3 volumes of ethanol were added. The reaction products were precipitated by brief centrifugation, and subjected to Sephadex G-25 gel filtration in 0.1 M NH4HCO3 to separate high molecular weight products from the remaining unreacted [35S]PAPS and degradation products.

For dermatan sulfate sulfotransferase assay, 0.05% protamine chloride instead of 0.005% protamine chloride was added in the same reaction mixture described above (26). For keratan sulfate sulfotransferase assay, the reaction mixture (50 µl) contained 50 mM imidazole-HCl, pH 6.4, 10 mM CaCl2, 2 mM dithiothreitol, 50 µg of keratan sulfate, 2 µM [35S]PAPS (about 5 × 105 cpm), and 25 µl of an enzyme solution (23). The reaction products were purified as described above. HNK-1ST activity was assayed using a synthetic acceptor, GlcAbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrowoctyl, as described previously (14, 18).

Chondroitin sulfate A (whale cartilage), chondroitin sulfate C (shark cartilage), completely desulfated and N-sulfated heparin, completely desulfated and N-acetylated heparin, and keratan sulfate were purchased from Seikagaku Corp. (Tokyo). Dermatan sulfate (porcine intestinal mucosa) was purchased from Calbiochem (San Diego, CA). Dermatan sulfate was subjected to chemical desulfation (27) before use as an acceptor. Desulfated dermatan sulfate produced Delta Di-0S in more than 95% of total unsaturated disaccharides after chondroitinase ABC digestion, confirming that more than 95% of sulfate group was removed.

Analysis of Enzymatic Reaction Products-- Enzymatic reaction products were analyzed after digestion with chondroitinase ABC (28) (Seikagaku Corp.), AC I Flavo (29) (Calbiochem), or chondroitinase B (Refs. 30 and 31, Calbiochem) and analyzed by HPLC or Bio-Gel P-4 gel filtration. Briefly, 35S-labeled products were digested with 25 milliunits of chondroitinase ABC for 1 h at 37 °C, 50 milliunits of chondroitinase AC I for 16 h at 37 °C, or 25 milliunits of chondroitinase B for 20 h at 30 °C. The resultant oligosaccharides were separated by HPLC using a Whatman Partisil SAX-10 column (4.6 mm × 25 cm) (Whatman, Clifton, NJ) equilibrated with 35 mM KH2PO4 at room temperature. The elution condition was modified from that published before (26). The column was eluted with a linear gradient from 35 mM KH2PO4 to 135 mM KH2PO4 in the first 20 min, then to 335 mM KH2PO4 in the next 20 min. Finally, the elution was linearly increased to 535 mM KH2PO4 in the additional 10 min. The column was then re-equilibrated with 35 mM KH2PO4. The flow rate was 1 ml/min and each fraction contained 0.5 or 1 ml. The products from [35S]sulfate-labeled dermatan sulfate were also applied to a column (10 mm × 120 cm) of Bio-Gel P-4 (Bio-Rad, Hercules, CA) equilibrated with 0.1 M NH4CH3CO2 as described previously (20).

The elution positions of Delta Di-0S, Delta Di-6S, Delta Di-4S, Delta Di-diSD, Delta Di-diSB, and Delta Di-diSE were determined at A232 nm. Various unsaturated disaccharides used as standards were purchased from Seikagaku Corp. Disaccharides or oligosaccharides released after chondroitinase digestion were subjected to chondro-6-sulfatase or chondro-4-sulfatase treatment as described before (28) (purchased from Seikagaku Corp.)

Northern Analysis-- Northern blots of multiple human tissues (CLONTECH) or human RNA Master BlotTM (CLONTECH) were hybridized with cDNA fragments isolated from pcDNA3.1-C4ST-1 and pcDNA3.1-C4ST-2 after 32P-labeling using a nick-translation kit (Prim-It·RmT) from Stratagene (La Jolla, CA).

Chromosome Mapping-- DNA samples were prepared from 83 radiation hybrids of human X rodent somatic cell hybrids containing human minichromosomes of the Stanford Human Genome Center G3 RH panel A (32, 33) (Research Genetics, Huntsville, AL). To determine the C4ST-1 and C4ST-2 loci, these DNA samples were analyzed by PCR. The PCR primers used to amplify the sequences corresponded to nucleotides 384-802 for C4ST-1 and nucleotides 528-797 for C4ST-2.

The PCR conditions were 10 cycles at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 30 s followed by 25 cycles at 94 °C for 1 min, 63 °C for 1 min, and 72 °C for 30 s. The maximum likelihood estimation was carried out by submitting the results to the RH server at the Stanford Genome Center and NCBI Gene Map '98, as described previously (33).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of cDNAs Encoding Chondroitin 4-O-Sulfotransferases (C4STs)-- Comparison of the amino acid sequences of cloned sulfotransferases demonstrated that there is a weak but discernible homologous sequence motif among Golgi-associated sulfotransferases (14). In particular, the RDP sequence motif was conserved among those sulfotransferases compared. By searching the EST data base for a novel cDNA related to HNK-1ST, two distinct cDNA sequences were found to have homology to the HNK-1ST sequence. The first cDNA (AA744877 in dbEST) encodes an open reading frame of 1,059 base pairs, predicting a protein of 352 amino acid residues (41,488 Da), which we subsequently termed C4ST-1 (Fig. 1). The second cDNA (AA182585 in dbEST) encodes an open reading frame of 1,245 base pairs, predicting a 414-amino acid residue protein (48,348 Da), which we subsequently termed C4ST-2 (Fig. 2). The cDNAs encoding C4ST-1 and C4ST-2 were cloned into pcDNA3.1/Hygro, resulting in pcDNA3.1-C4ST-1 and pcDNA3.1-C4ST-2, respectively.


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  		Fig. 1.   Nucleotide and translated amino acid sequences of C4ST-1. The signal/membrane anchoring domain is underlined and potential N-glycosylation sites are marked with closed circles.


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  		Fig. 2.   Nucleotide and translated amino acid sequences of C4ST-2. The signal/membrane anchoring domain is denoted by an underline and potential N-glycosylation sites are marked with closed circles.

The comparison of the amino acid sequences of C4ST-1 and C4ST-2 with HNK-1ST reveals the following points (Fig. 3). The sequences of the cytoplasmic segment and the transmembrane/anchoring domain are not strongly similar among these sulfotransferases, while the sequences are highly homologous to each other in the catalytic domains. There are four regions where sulfotransferases are highly homologous. The first two are the 5'-phosphosulfate-binding and 3'-phosphate-binding sites, respectively (Fig. 3). The third and fourth regions (A and B in Fig. 3) have not been reported before, but probably corresponds to two alpha -helical domains near the carboxyl-terminal ends (34).


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  		Fig. 3.   Comparison of amino acid sequences of HNK-1ST, C4ST-1, and C4ST-2 using the Clustal W program. Introduced gaps are shown as hyphens and aligned identical residues are boxed (black for all sequences, dark gray for two sequences). Putative binding sites for 5'-phosphosulfate group (5'-PSB) and 3'-phosphate group (3'-PB), and two highly conserved sequences (A and B) are denoted.

As a whole, the amino acid sequence of C4ST-1 is more homologous to that of C4ST-2 (41.8% identity) than that of HNK-1ST (31.6% identity), while HNK-1ST and C4ST-2 share 30.7% identity. None of the other amino acid sequences in the data base showed significant homology to these three sulfotransferases (see also "Discussion"). In fact, HNK-1ST, C4ST-1, and C4ST-2 share only 13% identity to the human C6ST (35).

Expression of C4ST-1 and C4ST-2-- To determine the acceptor specificity of C4ST-1 and C4ST-2, pcDNA3.1-C4ST-1, pcDNA3.1-C4ST-2, and control pcDNA3.1 were separately transfected into CHO cells. Sulfotransferase activity in cell extracts from the transfected cells was determined using various acceptors. First, neither C4ST-1 nor C4ST-2 exhibited activity toward the HNK-1 precursor acceptor GlcAbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrowoctyl. In contrast to HNK-1ST, C4ST-1 and C4ST-2 failed to express the HNK-1 antigen when transiently expressed in Lec2 cells containing HNK-1 precursor acceptor, as described previously (14) (data not shown). In addition, neither C4ST-1 nor C4ST-2 increased [35S]sulfate incorporation into NCAM- or CD34-human IgG chimeric protein in the presence or absence of beta 1,3-glucuronyltransferase (25) or core 2 beta 1,6-N-acetylglucosaminyl transferase (36) carried out as described previously (20) (data not shown). We then tested various glycosaminoglycans as acceptors. As shown in Fig. 4, C4ST-1 and C4ST-2 incorporated [35S]sulfate to chondroitin, chondroitin 4-O-sulfate (chondroitin sulfate A), chondroitin 6-O-sulfate (chondroitin sulfate C), and desulfated dermatan sulfate. In contrast, C4ST-1 and C4ST-2 did not incorporate [35S]sulfate to dermatan sulfate, desulfated and N-sulfated heparin, or desulfated and N-acetylated heparin or keratan sulfate (lower figures in Fig. 4).


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  		Fig. 4.   Incorporation of [35S]sulfate into various acceptors by C4ST-1, C4ST-2, C6ST, and HNK-1ST. Full-length C4ST-1, C4ST-2, C6ST, and HNK-1ST were transiently expressed in CHO cells and cell lysates from the transfected CHO cells were used as an enzyme source. Except for measuring HNK-1ST activity, all reaction mixtures were precipitated by ethanol and applied to a column of Sephadex G-25 and radioactivity eluted at the void volume was taken as an incorporated radioactivity. Acceptors used were chondroitin (C), chondroitin sulfate A (CA), chondroitin sulfate C (CC), dermatan sulfate (chondroitin sulfate B) (DS), and desulfated dermatan sulfate (D), completely desulfated and N-acetylated heparin (CDSNAc), completely desulfated and N-sulfated heparin (CDSNS), and keratan sulfate (KS). The radioactivity derived from the reaction without adding any acceptor is denoted as a minus (-).

When the activity of C6ST was assayed, C6ST incorporated [35S]sulfate into chondroitin, chondroitin sulfate A, chondroitin sulfate C, and keratin sulfate, as expected (22). On the other hand, HNK-1ST did not show any detectable activity toward these glycosaminoglycan acceptors (Fig. 4). These results indicate that newly cloned C4ST-1 and C4ST-2 are sulfotransferases that add sulfate(s) to chondroitin, chondroitin sulfate, and desulfated dermatan sulfate.

Identification of Reaction Products-- The above results showed that both C4ST-1 and C4ST-2 utilized almost identical acceptors, but did not show if C4ST-1 and C4ST-2 added sulfate to the 4- or 6-position of N-acetylgalactosamine or the 2-position of D-glucuronic acid.

To determine the structures of the sulfated products derived from chondroitin, we took advantage of the fact that isomers of sulfated disaccharide units produced by chondroitinase ABC can be separated by SAX-10 HPLC. As shown in Fig. 5A, almost all of the products by C4ST-1 eluted at the position of Delta Di-4S. The peak corresponding to Delta Di-4S released sulfate after treatment with chondro-4-sulfatase (Fig. 5B), but not with chondro-6-sulfatase (Fig. 5C). These results, combined together, indicate that C4ST-1 incorporated a sulfate to the 4-position of N-acetylgalactosamine in chondroitin. The products derived from chondroitin sulfate A or C showed a prominent peak corresponding to Delta Di-4S after chondroitinase ABC digestion, but did not contain any disulfated disaccharide (Fig. 5D), indicating that C4ST-1 adds a sulfate group only when neither glucuronic acid nor N-acetylgalactosamine in the acceptors contain a sulfate group. The amount of Delta Di-6S was almost the same as that observed in control experiments, indicating that 6-O-sulfation was due to an endogenous enzyme (Fig. 5A). The products from C4ST-2 were analyzed in an identical manner. The results are very similar to those described for C4ST-1 (Fig. 5, E-H).


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  		Fig. 5.   HPLC separation of chondroitinase ABC digests of 35S-labeled glycosaminoglycans obtained after incubation with [35S]PAPS and C4ST-1 or C4ST-2. A-C and E-G, 35S-labeled chondroitin after incubation with C4ST-1 (A-C) or C4ST-2 (E-G) were digested with chondroitinase ABC (A and E) and subjected to HPLC under the conditions described under "Experimental Procedures" (closed circles). Open circles denote the radioactivity obtained from 35S-labeled chondroitin incubated with the cell extracts from mock transfectants. Chondroitinase ABC-treated products were digested further with chondro-4-sulfatase (B and F) or chondro-6-sulfatase (C and G). D and H, similarly, 35S-labeled chondroitin sulfate C after incubation with C4ST-1 (D) or C4ST-2 (H) was digested with chondroitinase ABC and analyzed by HPLC. The arrows indicate the elution positions of: 0, Delta Di-0S (D-gluco-4-enepyranoside beta 1-3GalNAc); 6, Delta Di-6S (D-gluco-4-enepyranoside beta 1-3GalNAc(6S)); 4, Delta Di-4S (D-gluco-4-enepyranoside beta 1-3GalNAc(4S)); D, Delta Di-diSD (2-sulfo-D-gluco-4-enepyranoside beta 1-3GalNAc(6S)); E, Delta Di-diSE (D-gluco-4-enepyranosidebeta 1-3GalNAc(4S, 6S); SO42-, free sulfate ion. Delta Di-diSB (2-sulfo-D-gluco-4-enepyranoside beta 1-3GalNAc(4S)) elutes at almost the same position as Delta Di-diSE. The concentration of KH2PO4 in the elution solution is shown by dotted line.

Sulfation of Dermatan Sulfate by C4ST-1 and C4ST-2-- Both C4ST-1 and C4ST-2 incorporated [35S]sulfate to desulfated dermatan sulfate (Fig. 4). To determine how C4ST-1 and C4ST-2 act on dermatan sulfate, 35S-labeled products obtained from desulfated dermatan sulfate were digested with chondroitinase ABC. HPLC analysis of the digested material showed that C4ST-1 produced Delta Di-4S, indicating that C4ST-1 added a sulfate to the 4-position of N-acetylgalactosamine in desulfated dermatan sulfate (Fig. 6A). No disulfated disaccharide was detected. Almost identical results were obtained for C4ST-2 (data not shown). To further delineate the acceptor specificity of C4ST-1, the 35S-labeled products were digested with chondroitinase AC I, which cleaves only N-acetylgalactosaminyl linkage to D-glucuronic acid. The results demonstrated that approximately one-fourth of the total radioactivity was released as Delta Di-4S and the rest eluted in later fractions (Fig. 6B). After digestion with chondro-4-sulfatase, the peak corresponding to Delta Di-4S disappeared and a prominent free sulfate ion peak appeared instead (Fig. 6C). However, no significant change in larger 35S-labeled oligosaccharides, eluted after 24.5 min, was observed, indicating that chondro-4-sulfatase did not release sulfate from oligosaccharides larger than disaccharides. Digestion of the same material by chondro-6-sulfatase, on the other hand, barely changed the elution profile (Fig. 6D), being consistent with the above conclusions that C4ST-1 incorporated [35S]sulfate to the 4-position of N-acetylgalactosamine.


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  		Fig. 6.   Analysis of 35S-labeled dermatan sulfate obtained after incubation with C4ST-1. A-D, HPLC analysis of 35S-labeled dermatan sulfate after digestion with chondroitinase ABC (A), chondroitinase AC I (B), chondroitinase AC I followed by chondro-4-sulfatase (C) or chondro-6-sulfatase (D) (closed circles). The elution positions of Delta Di-0S (0), Delta Di-4S (4), Delta Di-6S (6), free sulfate ion (SO42-), Delta Di-diSD (D), and Delta Di-diSE (E) are shown. In A and B, open circles denote the radioactivity obtained from mock experiments. E and F, Bio-Gel P-4 gel filtration analysis of 35S-labeled dermatan sulfate after digestion with chondroitinase AC I followed by chondro-6-sulfatase (E) or digestion with chondroitinase B followed by chondro-6-sulfatase (F). The elution positions of free sulfate ion (SO42-), Delta Di-4S (4), tetrasaccharides (A), and hexasaccharides (B) obtained after digestion of chondroitin sulfate A with chondroitinase ABC, and void volume (Vo) are shown.

To determine the nature of larger oligosaccharides obtained after chondroitinase AC I treatment, the same sample analyzed in Fig. 6D was subjected to Bio-Gel P-4 gel filtration. The results showed that approximately one-fourth of the total radioactivity eluted at Delta Di-4S and approximately 10% of total radioactivity eluted as tetrasaccharide and hexasaccharide (Fig. 6E). Chondroitinase AC I can release Delta Di-4S only from GlcAright-arrowGalNAc(4S) that is flanked by GlcAright-arrowGalNAc units (29). These results suggest that [35S]sulfate was incorporated into GlcAright-arrowGalNAc unit.

To corroborate the above experiments, intact [35S]sulfate-labeled dermatan sulfate was directly digested by chondroitinase B, which cleaves a sulfated N-acetylgalactosaminyl linkage to iduronic acid flanked by IdoAright-arrowGalNAc units (30, 31). The results demonstrated no release of 35S-labeled Delta Di-4S or 35S-labeled oligosaccharides (Fig. 6F). These results combined together indicate that C4ST-1 and most likely C4ST-2 preferentially incorporate a sulfate at the 4-position of N-acetylgalactosamine in GlcAright-arrowGalNAc than in the IdoAright-arrowGalNAc unit.

C4ST-1 and C4ST-2 Are Differentially Expressed in Various Tissues-- To determine the expression of C4ST-1 and C4ST-2 transcripts in various tissues, Northern and dot blot analysis was carried out. Gel fractionated blot (Fig. 7) and dot blot (Fig. 8) analyses show that the C4ST-1 transcript is highly expressed in spleen, thymus, peripheral blood leukocytes, lymph node, bone marrow, lung, and placenta. In contrast, the transcripts of C4ST-2 are expressed more ubiquitously (Fig. 7), but significantly more in spinal cord, heart, thyroid, pituitary gland, adrenal gland, small intestine, spleen, peripheral blood leukocytes, thymus, lung, fetal kidney, fetal spleen, and fetal lung on the dot blot (Fig. 8). These results show that C4ST-1 is mostly expressed in leukocytes and hematopoietic tissues, while C4ST-2 is widely expressed in various tissues, including endocrine organs and nervous systems.


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  		Fig. 7.   Northern analysis of C4ST-1 and C4ST-2 transcripts. Each lane contained 2 µg of poly(A)+ RNA. The blots were hybridized with the appropriate 32P-labeled C4ST cDNAs. Each blot contained four or eight lanes and was run separately. The migration positions of molecular markers are shown at the left. The positions of the transcripts are indicated by arrowheads.


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  		Fig. 8.   Dot blot analysis of C4ST-1 and C4ST-2 transcripts. Human RNA Master BlotTM shown at the far left was sequentially hybridized to 32P-labeled human C4ST-1 or C4ST-2 cDNA.

Chromosomal Mapping of the C4ST-1 and C4ST-2 Genes-- To determine the chromosomal localization of C4ST-1 and C4ST-2 genes, PCR analysis was carried out using the Stanford G3 RH panel. PCR primers were chosen from coding regions of C4ST-1 and C4ST-2 genes, and based on the criteria that PCR products showed the same molecular weight when C4ST-1 or C4ST-2 cDNA or genomic DNA was used as a template, but not using hamster genomic DNA. This analysis placed C4ST-1 between D12S1607 and D12S360, thus mapping the gene to the q23 region of chromosome 12. Similarly, the C4ST-2 gene was placed between D7S2563 and D7S2521, mapping the gene to the p22 region of chromosome 7.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study describes the isolation of novel cDNAs encoding chondroitin 4-O-sulfotransferase by searching the EST data bases for cDNAs homologous to the human HNK-1ST (14). HNK-1ST adds a sulfate to the 3-position of glucuronic acid, which in turn is attached to the 3-position of galactose in N-acetyllactosamine. C4ST, on the other hand, adds a sulfate to the 4-position of N-acetylgalactosamine, which is in turn attached to the 4-position of glucuronic acid. These results are striking since these two acceptors are rather dissimilar. The beta -glucuronyl residue in HNK-1 glycan is at the nonreducing terminal. In contrast, C4ST apparently acts on an already elongated chondroitin chain since no preferential addition to shorter acceptors has been noticed when the products were analyzed by gel filtration (data not shown, see also Ref. 26). The hydroxyl groups in both C-3 of glucuronic acid and C-4 of N-acetylgalactosamine are projected above their respective pyranose rings in their normal conformations (37). It is tempting to speculate that the active sites of both HNK-1ST and C4ST may approach the acceptor from above the plane of GlcAbeta 1right-arrow3Galbeta 1right-arrow4GlcNAcbeta 1right-arrowR (for HNK-1ST) and GlcAbeta 1right-arrow3GalNAcbeta 1right-arrow4GlcAbeta 1right-arrowR (for C4ST).

It is noteworthy that C4ST-1 and C4ST-2 share only 41.8% identity at the amino acid levels, but share a common catalytic property. C4ST-1 and C4ST-2, however, are much more homologous to each other in the vicinity of 5'-phosphosulfate and 3'-phosphate binding sites (Fig. 3). Moreover, C4ST-1 and C4ST-2 apparently share common structural domains toward the carboxyl-terminal regions (A and B in Fig. 3). These regions do not share homology with other sulfotransferases (34) and further studies are necessary to determine their roles.

Fig. 9 illustrates the phylogenetic relationship of cloned Golgi-associated sulfotransferases that add a sulfate on carbohydrate acceptors. The results clearly indicate that C4ST-1, C4ST-2, and HNK-1ST form a gene family distinct from the rest of the sulfotransferase gene families. The members within the same gene family depicted in Fig. 9 catalyze identical or similar reactions, except for one case. LSST, I-GlcNAc6ST, GlcNAc6ST, C6ST, and KSST form a gene family whose acceptor specificities are not clearly related to each other. The cDNAs (GlcNAc6ST, LSST, and I-GlcNAc6ST) encoding a sulfotransferase that adds a sulfate to the 6-position of N-acetylglucosamine at the nonreducing terminal were identified in EST data base for their homology to C6ST or KSST (19-21, 38). In contrast, C6ST and KSST add a sulfate on the 6-position of N-acetylgalactosamine or galactose on already elongated substrates (22, 23). These results, combined together with the results obtained in the present study, indicate that it is possible to identify cDNAs encoding enzymes that utilize very different acceptors from those utilized by a protein whose cDNA was used as a probe. Further studies will be significant to determine the three-dimensional structures of these sulfotransferases bound to acceptors in order to test if these enzymes approach their acceptors from above the plane of the acceptors.


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  		Fig. 9.   Schematic representation of phylogenetic tree of Golgi-associated carbohydrate sulfotransferases. Amino acid sequences predicted from cloned cDNAs are compared using the Clustal W method with PAM250 residue weight table. The following sequences are compared: human heparan sulfate D-glucosaminyl 3-O-sulfotransferase, hu HS3OST-1 (53), -2, -3A, and -3B (54); human heparan sulfate N-deacetylase/sulfotransferase, hu HSNDST-1, -2, and -3 (55-58); human chondroitin D-N-acetylgalactosamine-6-O-sulfotransferase, hu C6ST (35); human keratan sulfate D-galactose-6-O-sulfotransferase, hu KSST (23); human D-N-acetylglucosamine-6-O-sulfotransferase, hu GlcNAc6ST (59); human intestinal D-N-acetylglucosamine-6-O-sulfotransferase, hu I-GlcNAc6ST (38); human L-selectin ligand sulfotransferase, hu LSST (20, 21); mouse L-selectin ligand sulfotransferase, mo LSST (20, 21); human HNK-1 sulfotransferase, hu HNK-1ST (14); human chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase, hu C4ST-1 and -2 (present study); human dermatan/chondroitin uronyl-2-O-sulfotransferase, hu CS/DS2OST (60); human heparan sulfate 2-O-sulfotransferase, hu HS2OST (61); human galactosylceramide D-Gal-3-O-sulfotransferase, hu GalCerST (62); mouse heparan sulfate D-sulfoglucosamine-6-O-sulfotransferase, mo HS6OST-1, -2, and -3 (63).

The present study demonstrated that both C4ST-1 and C4ST-2 act much more efficiently on non-sulfated chondroitin or desulfated dermatan sulfate than chondroitin sulfate A, chondroitin sulfate C, or dermatan sulfate (Fig. 4). No disulfated disaccharide was released after chondroitinase ABC digestion of reaction products derived from chondroitin sulfate C (Fig. 5). These results indicate that C4ST-1 and C4ST-2 add sulfate only on unsulfated N-acetylgalactosamines.

The present study also demonstrated that C4ST-1 and C4ST-2 add a sulfate to the 4-position of N-acetylgalactosamine residues in dermatan sulfate, which had been chemically desulfated (Figs. 4 and 6). The detailed analysis of dermatan sulfated by C4ST-1 revealed the following points. Even though glucuronic acid residues are minor components in the dermatan sulfate, at least one-fourth of the total radioactivity was detected in Delta Di-4S when released by chondroitinase AC I digestion. In this case, Delta Di-4S was released only when 4-sulfated N-acetylgalactosamine are positioned between two glucuronic acids. This finding suggests that C4ST-1 acts on N-acetylgalactosamine residues next to glucuronic acid. If C4ST-1 transfers a sulfate to N-acetylgalactosamine linked to iduronic acid as efficiently as to N-acetylgalactosamine linked to glucuronic acid, more 35S-labeled oligosaccharides would be released by chondroitinase B digestion than by chondroitinase AC I digestion. However, Delta Di-4S was hardly released after chondroitinase B digestion (Fig. 6). These results, combined together, indicate that the GlcAright-arrowGalNAc unit is a much better acceptor for C4ST-1 (and most likely for C4ST-2 as well) than the IdoAright-arrowGalNAc unit.

The results obtained in the present study are similar to those obtained on C4ST purified from a rat chondrosarcoma cell line (26). However, the C4ST in that study added a sulfate more on desulfated dermatan sulfate (porcine skin) and the products were highly susceptible to chondroitinase AC II, which cleaves only a GlcAright-arrowGalNAc unit flanked by GlcAright-arrowGalNAc (39). This discrepancy is probably due to the difference in the source of dermatan sulfate and that the dermatan sulfate from pig skin probably contains more glucuronic acid residues which are clustered than the dermatan sulfate from porcine intestinal mucosa used in the present study.

The detailed biosynthetic steps of dermatan sulfate are currently unknown. Malström (40) showed that epimerization from glucuronic acid to iduronic acid takes place in unsulfated chondroitin. However, the conversion to iduronic acid in that report was only 15%, which is much lower than the actual iduronic acid content in nature (41). Moreover, this epimerization is reversible for unsulfated forms, but IdoAright-arrowGalNAc(4S) is not converted to GlcAright-arrowGalNAc(4S). On the other hand, Silbert et al. (42) showed that lower sulfation leads to lower epimerization, suggesting that sulfation at the 4-position of N-acetylgalactosamine precedes epimerization. If C4ST-1 and C4ST-2 involve mainly in the biosynthesis of dermatan sulfate, the expression of C4ST-1 or C4ST-2 should result in higher expression of dermatan sulfate containing large amounts of IdoAright-arrowGalNAc(4S). However, if the same enzyme contributes to form both chondroitin sulfate and dermatan sulfate, several factors may determine the destination of these synthesized molecules to chondroitin sulfate or dermatan sulfate. When an epimerase is expressed, it converts a glucuronic acid to an iduronic acid, and this reaction is accelerated by the sulfation of N-acetylgalactosamine introduced by C4ST. In contrast, in the absence of an epimerase, no dermatan sulfate is formed. Thus, the presence of an epimerase should be a main regulator, but another possibility needs to be considered. The results of in vitro enzyme assay showed that the sulfotransferase activity of C4ST to chondroitin and dermatan reciprocally changes depending upon the concentration of protamine chloride in the reaction mixture (Ref. 26 and the present study). The concentration of protamine chloride also affects the activity of chondroitin 6-O-sulfotransferase including its substrate specificity (43). These findings suggest that the environmental factors affecting the activity of C4ST might contribute to the regulation of the chondroitin sulfate and dermatan sulfate biosynthesis. It is also possible that another sulfotransferase preferentially acting on IdoAright-arrowGalNAc is involved in dermatan sulfate biosynthesis. Further studies will be significant to clarify these points.

While we were preparing this manuscript, the mouse counterpart of C4ST-1 was reported (44). The human and mouse C4ST-1 have 96.0% identity at the amino acid levels. The expression profile of the mouse C4ST is slightly different from that of human C4ST-1 in that it is mainly expressed in the brain and kidney. The mouse C4ST-1, however, apparently exhibits almost the same substrate specificity as the human C4ST-1 and C4ST-2 (44).

The transcripts of human C4ST-1 and C4ST-2 are differentially expressed in various tissues. The C4ST-1 transcript is predominantly expressed in peripheral blood leukocytes and hematopoietic tissues such as, bone marrow and spleen, while the C4ST-2 transcript is more widely expressed, including in the pituitary gland, adrenal gland, spinal cord, small intestine, spleen, and lung (Fig. 7 and 8). These results indicate that C4ST-1 and C4ST-2 may play complementary roles in different tissues.

Chondroitin sulfate proteoglycans have been found in the brain and have been shown to play roles in neural cell adhesion and neurite outgrowth (45-48), and neural cell migration (49). Chondroitin sulfate is also present in blood cells and has been shown to be involved in interaction with CD44 (50, 51) and L-selectin (52). C4ST-1 and C4ST-2 cloned in the present study will be powerful tools to determine the roles of chondroitin sulfate in these various biological systems.

    ACKNOWLEDGEMENTS

We thank the members of our laboratories for useful discussions, and Susan Wynant and Risa Tabata for organizing the manuscript.

    FOOTNOTES

* This work was supported by National Cancer Institute Grants P01CA71932 and CA33895.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper has been submitted to GenBank with accession number AF239820 for C4ST-1 and AF239822 for C4ST-2.

Dagger Present address: Graduate School of Science, University of Hokkaido, Sapporo, 060-0810 Japan.

§ To whom correspondence should be addressed: The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3144; Fax: 858-646-3193; E-mail: minoru@burnham.org.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002443200

    ABBREVIATIONS

The abbreviations used are: GlcA, D-glucuronic acid; IdoA, L-iduronic acid; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; EST, expressed sequence tag; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; HNK-1ST, HNK-1 sulfotransferase; C4ST, chondroitin 4-O-sulfotransferase; C6ST, chondroitin 6-O-sulfotransferase; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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