Characterization of a Heparan Sulfate 3-O-Sulfotransferase-5, an Enzyme Synthesizing a Tetrasulfated Disaccharide*

Hideo Mochizuki {ddagger} §, Keiichi Yoshida ¶, Masanori Gotoh {ddagger} ||, Shigemi Sugioka {ddagger}, Norihiro Kikuchi {ddagger} **, Yeon-Dae Kwon {ddagger} **, Akira Tawada §, Kennichi Maeyama §, Niro Inaba {ddagger} {ddagger}{ddagger}, Toru Hiruma {ddagger} §§, Koji Kimata ¶¶ and Hisashi Narimatsu {ddagger} ||||

From the {ddagger}Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8586, Japan, the §Central Research Laboratories, Seikagaku Corporation, 3-1253 Tateno, Higashi-yamato, Tokyo 207-0021, Japan, the Mizutani Foundation for Glycoscience, Sen-i Kaikan, 3-1-11 Nihonbashi-honcho, Chuo-ku, Tokyo 103-0023, Japan, ||Amersham Biosciences KK, 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan, **Mitsui Knowledge Industry Co., Ltd., 1 Honcho, Nakano-ku, Tokyo 164-8721, Japan, {ddagger}{ddagger}JGS Japan Genome Solutions, Inc., 51 Komiya-cho, Hachioji, Tokyo 192-0031, Japan, the §§Frontier Research Division, Fundamental Research Department, Fujirebio, Inc., 51 Komiya-cho, Hachioji, Tokyo 192-0031, Japan, and the ¶¶Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan

Received for publication, February 21, 2003 , and in revised form, April 22, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparan sulfate D-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912–37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as {Delta}HexA-GlcN(NS,3S,6S) and {Delta}HexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of {Delta}HexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparan sulfate proteoglycans (HSPGs)1 are ubiquitously present on the cell surface and in the extracellular matrix and have divergent structures and functions (13). Many biological functions of HSPGs are mediated by interactions between the heparan sulfate chain and a variety of proteins, including protease inhibitors, heparin-binding growth factors, extracellular matrix components, protease, and lipoprotein lipase (48). Moreover, some pathogens exploit heparan sulfate of the host cell surface, which binds to coat proteins or cell surface proteins of pathogens, at the invasion (9, 10). Most of the interactions between heparan sulfate and various functional proteins occur in certain regions of the heparan sulfate chain with specific sulfated monosaccharide sequences such as binding sites for antithrombin III (AT III) (11), acidic fibroblast growth factor (12), basic fibroblast growth factor (1316), and hepatocyte growth factor (17, 18). The ability of cells to produce heparan sulfate with such sequences depends on the specific mechanisms of heparan sulfate biosynthesis (19, 20). Heparan sulfate is initially synthesized as a polymer of a disaccharide repeat sequence, -glucuronic acid-{beta}1,4-N-acetylglucosamine-{alpha}1,4-. This polymer is then N-deacetylated/N-sulfated and subsequently undergoes epimerization of glucuronic acid (GlcA) to iduronic acid (IdoA), 2-O-sulfation of uronic acid, and 6-O-sulfation of glucosamine residues. Additionally, a rare but functionally important modification, 3-O-sulfation of the glucosamine residue, also occurs (21). Most of the enzymes involved in the biosynthesis of heparan sulfate have been purified and cloned (22, 23). Some of them have been shown to be present as isoforms. These isoforms play important roles in generating the specific and diverse structures of heparan sulfate. Four isoforms of N-deacetylase/N-sulfotransferase have been reported. These isoforms are distinguishable from each other with respect to the extent of N-sulfation of heparan sulfate, the ratio of N-deacetylase to N-sulfotransferase activities and the expression pattern in various tissues (2426). Three isoforms of heparan sulfate 6-O-sulfotransferase were found to have different specificities and different expression patterns (27). Six isoforms of 3-O-sulfotransferase (3-OST) with different spectra of acceptor substrate specificity have also been reported. 3-OST-1 transfers sulfate to GlcA-GlcN(NS,±6S) and produces the GlcA-GlcN(NS,3S,±6S) unit essential to the AT III binding domain (28, 29). 3-OST-2 transfers sulfate to GlcA/IdoA(2S)-GlcN(NS) units (30). 3-OST-3A transfers sulfate to N-unsubstituted glucosamine residues and forms IdoA(2S)-GlcN(3S,±6S) units (31). 3-OST-3B has a sulfotransferase domain 99.2% identical to that of 3-OST-3A and also forms identical reaction products (21). 3-OST-3-modified heparan sulfate specifically binds to glycoprotein D (gD) of herpes simplex virus type-1 (HSV-1) and also makes cells susceptible to HSV-1 entry (32, 33). The substrate specificity of 3-OST-4 has not been reported (34). 3-OST-5 was recently cloned and its reaction products were identified as GlcA-aMan(3S,6S), IdoA(2S)-aMan(3S) and IdoA(2S)-aMan(3S,6S) by nitrous acid degradation. Interestingly, 3-OST-5-modified heparan sulfate binds to AT III and gD (35).

In this study, we cloned human 3-OST-5, and focused on the characterization of reaction products. Our results indicate novel substrate specificities and tissue distribution of 3-OST-5. Finally, we demonstrated the novel tetrasulfated disaccharide structure in natural heparan sulfate.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[35S]PAPS was purchased from PerkinElmer Life Sciences (Boston, MA). Sodium [3H]borohydride was obtained from ICN Biomedicals (Irvine, CA). Heparinase, heparitinase I, heparitinase II, an unsaturated glucosaminoglycan disaccharide kit, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, hyaluronic acid, and Cellulofine GCL-25-sf were from Seikagaku Corporation (Tokyo, Japan). Heparin from porcine intestinal mucosa, D-glucosamine N-3-disulfate (GlcN(NS,3S)), and D-glucosamine N-3,6-trisulfate (GlcN(NS,3S,6S)) were from Sigma. The TSKgel G2500PW column was from Tosoh (Tokyo, Japan). The CarboPacTM PA1 column was from Dionex (Sunnyvale, CA). The SuperdexTM Peptide HR10/30 column was from Amersham Biosciences (Amersham Place, UK). BioGel P-4 was from Bio-Rad Laboratories (Hercules, CA). Heparan sulfate from bovine kidney and from bovine intestine and 2-sulfatase, {Delta}4,5-glycuronate-2-sulfatase from Flavobacterium heparinum, were provided by Seikagaku Corporation.

Construction and Purification of 3-OST-5 Protein Fused with FLAG Peptide—The putative catalytic domain of 3-OST-5 (amino acids 63–346) was cloned and expressed as a secreted protein fused with a FLAG peptide in insect cells according to the instruction manual of GATEWAYTM Cloning Technology (Invitrogen, Groningen, Netherlands). Because the catalytic domain is encoded by a single exon (35), an ~1-kb DNA fragment containing the entire exon II was amplified by PCR using the human genomic DNA (Clontech), as a template, and two primers, 5'-ACTGGGGAACCAGAAAAATGAAAAG-3' and 5'-GTGTCTCCAGGCACAACACATAGTG-3'. The PCR product was used as a template in a nested PCR to amplify the DNA fragment containing the putative catalytic domain. The forward primer was 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTTTAAGCGTGGCCTGCTGCACGAG-3' and the reverse primer was 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTAGGGCCAGTTCAATGTCCTCCC-3'. The amplified fragment was cloned into a pDONRTM 201 vector (Invitrogen) and subsequently cloned into a vector pFBIF linearized by NcoI to yield pFBIF containing the catalytic domain of 3-OST-5 (pFBIF-3OST). pFBIF is an expression vector derived from pFastBac1 (Invitrogen) and contains a fragment encoding a signal peptide of human immunoglobulin (MHFQVQIFSFLLISASVIMSRG), the FLAG peptide (DYKD-DDDK) and the conversion site for the GATEWAYTM system. DH10BAC competent cells (Invitrogen) were transformed with the pFBIF-3OST and then bacmid DNA was prepared from the cells. Recombinant virus was prepared according to the instruction manual of BAC-TO-BAC Baculovirus Expression Systems (Invitrogen). Sf21 insect cells (BD Pharmingen, San Diego, CA) were infected with the recombinant virus and incubated at 27 °C until the survival rate was less than 50% to yield secreted recombinant 3-OST-5 proteins fused with the FLAG peptide. The secreted enzyme was purified using anti-FLAG M1 monoclonal antibody agarose affinity gel (Sigma). The culture media and affinity gel mixed overnight at 4 °C were centrifuged for 5 min, and the supernatant was aspirated. The affinity gel was washed twice with 50 mM TBS(50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 1 mM CaCl2, and resuspended in 50 mM TBS to obtain a 50% slurry. The immobilized enzyme was stable at –80 °C for at least six months.

Assay for Sulfotransferase Activity—The standard reaction mixture contained 50 mM imidazole-HCl, pH 6.8, 75 µg/ml of protamine chloride, 0.5 mM (as hexosamine) acceptor substrates, 1.5 µM [35S]PAPS (about 5 x 105 dpm), and 1 µl of immobilized enzyme in a final volume of 50 µl. Acceptor substrates used to examine the substrate specificity were heparan sulfate, heparin, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, and hyaluronic acid. After incubation at 37 °C for 20 min, the reaction was stopped by heating at 100 °C for 1 min. The reaction mixture was filtrated with an Ultrafree-MC (MILLIPORE, Bedford, MA) and 60 µg of chondroitin sulfate C was added as a carrier. 35S-Labeled substrates were precipitated with 3 volumes of ethanol containing 1.3% potassium acetate and separated completely from [35S]PAPS and its degradation products by gel filtration HPLC on a TSKgel G2500PW column (0.75 x 30 cm) equilibrated with 0.2 M NaCl at a flow rate of 0.6 ml/min and the column temperature of 35 °C. Fractions of 0.3 ml were collected and the radioactivity was determined by liquid scintillation counting.

Preparation of Oligosaccharides from Heparin—It was reported that the heparinase digestion of heparin produces about 45% each disaccharide and tetrasaccharide, and a few percent hexasaccharide (36, 37). Two milligrams of heparin was digested with 0.2 units of heparinase and applied to a BioGel P-4 column (1.5 x 140 cm) equilibrated with 0.1 M ammonium bicarbonate. Elution positions of each oligosaccharide were monitored at 232 nm. Disaccharide, tetrasaccharide and hexasaccharide fractions were pooled and desalted by lyophilization.

HPLC Analysis on an Anion-exchange Column and a Gel Filtration Column—Anion-exchange (SAX) separation was performed on a CarboPacTM PA1 column (4 x 250 mm) as described (38). A combination of five linear LiCl gradients was used, from 30 to 180 mM (0–5 min), from 180 to 570 mM (5–8 min), from 0.57 to 1.14 M (8–15 min), from 1.14 to 2.1 M (15–20 min), and from 2.1 to 2.28 M (20–28 min) followed by 2.28 M (from 28 min). The flow rate was 0.8 ml/min, and the column temperature was 40 °C. Fractions were collected and the radioactivity was determined by liquid scintillation counting. To determine the molecular size of oligosaccharides, gel filtration HPLC was performed on a SuperdexTM Peptide HR10/30 column (1 x 30 cm, x 2 columns in series) as described (39). The column was equilibrated with 0.2 M NaCl at a flow rate of 0.8 ml/min, at room temperature.

Preparation of 35S-Labeled Heparan Sulfate and Digestion with Heparin Lyases—One milligram of heparan sulfate from bovine kidney and [35S]PAPS (6.6 x 107 dpm) was incubated with 0.5 ml of standard reaction mixture containing 67 µl of immobilized enzyme at 37 °C for 3 h. After the incubation, the reaction mixture was filtrated, and 35S-labeled heparan sulfate was precipitated with ethanol as described above. The precipitate was dried at room temperature. The 35S-labeled heparan sulfate was digested with a mixture of 0.5 units of heparinase, 0.3 units of heparitinase I, and 0.2 units of heparitinase II in 0.3 ml of 20 mM sodium acetate buffer (pH 7.0) containing 2 mM calcium acetate at 37 °C for 2 h. The reaction was stopped by heating at 100 °C for 1 min, and the mixture was filtrated. The digested products were subjected to chromatography on a BioGel P-4 column (1.5 x 140 cm) equilibrated with 0.1 M ammonium bicarbonate at a flow rate of 5 ml/h. Fractions of 2 ml were collected. The fractions indicated by horizontal bars in Fig. 2, named P4-1 and P4-2, were pooled, lyophilized and completely desalted with a gel filtration column.



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FIG. 2.
Gel filtration chromatography on a BioGel P-4 column of 35S-labeled heparan sulfate digested with a mixture of heparin lyases. 35S-Labeled heparan sulfate digested with a mixture of heparin lyases was applied to a BioGel P-4 column as described under "Experimental Procedures." Fractions of 2 ml were collected, and radioactivity was determined. The fractions indicated by horizontal bars, named P4-1 and P4-2, were pooled.

 

Digestion with 2-Sulfatase—The 35S-labeled disaccharides were digested with 4 milliunits of 2-sulfatase in 20 mM sodium acetate buffer (pH 6.5) containing 1.5 mg/ml of bovine serum albumin. After incubation at 37 °C for 2 h, the reaction was stopped by heating at 100 °C for 1 min. The digested products were analyzed by HPLC on a SAX column as described above.

Determination of Glucosamine Residue—The 35S-labeled disaccharides were treated with mercuric acetate to remove unsaturated uronic acid residues as described (40). The disaccharide fraction was mixed with an equal volume of 70 mM mercuric acetate (pH 5.0) and incubated at room temperature for 10 min. The reaction products, [35S]sulfated glucosamines, were reduced with sodium borohydride as described (41) and analyzed by HPLC on a SAX column as described above. GlcN(NS,3S) and GlcN(NS,3S,6S) were reduced with sodium [3H]borohydride and used as a standard.

Preparation of 35S-Labeled Heparin and Digestion with Heparin Lyases—35S-Labeled heparin was prepared by incubating 0.3 mg of heparin with [35S]PAPS (2.3 x 107 dpm) and 20 µl of immobilized enzyme in a standard reaction mixture. The 35S-labeled heparin was precipitated with ethanol and digested with a mixture of heparin lyases as described above. The digested products were subjected to HPLC on a SAX column as described above. Fractions of 0.24 ml were collected and the radioactivity was determined. A peak fraction at 30.1 min (indicated by a white arrow in Fig. 6) was applied to a Cellulofine G-25-sf column (1 x 28 cm) equilibrated with distilled water to remove LiCl. Radioactive fractions were pooled and concentrated under vacuum.



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FIG. 6.
HPLC analysis on a SAX column of 35S-labeled products derived from heparin. 35S-Labeled heparin produced by the sulfotransferase reaction of 3-OST-5 was digested with a mixture of heparin lyases as described under "Experimental Procedures." The digested products were separated by HPLC on a SAX column. The conditions for HPLC were as described under "Experimental Procedures." Fractions of 0.24 ml were collected, and radioactivity was determined. The arrows indicate the elution positions of {Delta}HexA(2S)-GlcN(NS,6S) ({Delta}Di-triS1), {Delta}HexA-GlcN(NS,3S,6S) ({Delta}Di-triS2), and {Delta}HexA(2S)-GlcN(NS,3S) ({Delta}Di-triS3). The fraction indicated by a white arrow was used for further analysis.

 

Quantitative Analysis of the 3-OST-5 Transcripts in Human Tissues by Real-time PCR—For quantification of 3-OST-5 transcripts, we employed the real-time PCR method, as described in detail previously (42). Total RNAs derived from various human tissues were purchased from Clontech and cDNAs were synthesized with the SuperScriptTM First-strand Synthesis System for RT-PCR (Invitrogen). To obtain the control DNA of 3-OST-5, a DNA fragment containing exon I and the 5'-terminal region of exon II was amplified by PCR using the Marathon ReadyTM cDNA, as a template, and two primers, 5'-TCTATCGCAGGCTGCAGCAGTCCT-3' and 5'-GGAACTCGTGCAGCAGGCCACGC-3'. Standard curves for the 3-OST-5 and the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs, were generated by serial dilution of the control DNA of 3-OST-5, and a pCR2.1 vector (Invitrogen) containing the GAPDH gene. The primer sets and the probes for 3-OST-5 were as follows: the forward primer was 5'-GCCAGAGTTGGGAGCTTGG-3', the reverse primer was 5'-ACCCAGTCGACCTTCAATGG-3', and the probe for 3-OST-5 was 5'-TAGGCTACAACCCATTTG-3' with a minor groove binder (43). PCR products were continuously measured with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The relative amounts of 3-OST-5 transcripts were normalized to the amount of GAPDH transcript in the same cDNA.

Analysis of Bovine Intestinal Heparan Sulfate—Bovine intestinal heparan sulfate (0.2 mg) was digested with a mixture of heparin lyases as described above. The digested products were reduced with sodium [3H]borohydride and subjected to HPLC on a SAX column as outlined above. 35S-Labeled tetrasulfated disaccharide obtained from 3-OST-5-modified heparin in this study was reduced with sodium borohydride and used as a standard.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Specificity for Acceptor Substrate—The putative catalytic domain of 3-OST-5 was cloned and expressed, and the sulfotransferase activity was examined as described in "Experimental Procedures." As expected, 3-OST-5 exhibited sulfotransferase activity toward heparan sulfate and heparin. On the other hand, it exhibited no activity toward chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate, and hyaluronic acid. The activity of 3-OST-5 for heparan sulfate is presented as 100%, and the other activities are given as relative values in Table I.


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TABLE I
Acceptor substrate specificity of 3-OST-5

Sulfotransferase activities were assayed using various glycosaminoglycans as acceptor substrates as described under "Experimental Procedures."

 

Digestion of 35S-Labeled Heparan Sulfate with Heparin Lyases—35S-Labeled heparan sulfate, which was prepared by incubating heparan sulfate with [35S]PAPS and the recombinant 3-OST-5, was digested with a mixture of heparin lyases and analyzed by HPLC on a SAX column as described under "Experimental Procedures." When monitored at 232 nm, six known disaccharide units of heparan sulfate, {Delta}HexA-GlcNAc, {Delta}HexA-GlcN(NS), {Delta}HexA-GlcNAc(6S), {Delta}HexA-GlcN(NS,6S), {Delta}HexA(2S)-GlcN(NS), and {Delta}HexA(2S)-GlcN(NS,6S), were identified (Fig. 1A). Several radioactive peaks were observed, but no peak was identified at the position of those six units (Fig. 1B). Therefore, the 35S-labeled products were different from these six disaccharides. The digestion products were applied to a gel filtration column of BioGel P-4, and two major 35S radioactive peaks were obtained (Fig. 2). The fractions indicated by a horizontal bar in Fig. 2, named P4-1 and P4-2, were pooled and desalted for further analysis. To confirm the molecular size, these two fractions were analyzed by HPLC on a gel filtration column. The elution profile of standard oligosaccharides prepared from heparin is shown in Fig. 3A. 35S-Labeled substances, P4-1 and P4-2 in Fig. 2, were eluted at the position of the tetrasaccharide and disaccharide, respectively (Fig. 3, B and C). These two fractions were analyzed by HPLC on a SAX column, and each fraction gave two components. Judging from the elution position, P4-1 and P4-2 corresponded to peaks 1 and 3, and peaks 2 and 4, respectively, which were four major peaks observed in Fig. 1B. To confirm the resistance to enzyme digestion, P4-1 was treated with the heparin lyase mixture again. No change was observed in the appearance of peak 1 and peak 3 during HPLC analysis on a SAX column. Moreover, this fraction is also resistant to nitrous acid degradation. The P4-1 was not characterized further, since these tetrasaccharides encountered some difficulty in further identification using enzymes (see "Discussion").



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FIG. 1.
HPLC analysis on a SAX column of 35S-labeled products derived from heparan sulfate. 35S-Labeled heparan sulfate produced by the sulfotransferase reaction of 3-OST-5 was digested with a mixture of heparin lyases as described under "Experimental Procedures." The digested product was analyzed by HPLC on a SAX column. The conditions for HPLC were as described under "Experimental Procedures." The absorbance was monitored at 232 nm (panel A). Fractions of 0.22 ml were collected, and radioactivity was determined by liquid scintillation counting (panel B). The arrows in panel A indicate the elution positions of {Delta}HexA-GlcNAc ({Delta}Di-0S), {Delta}HexA-GlcN(NS) ({Delta}Di-NS), {Delta}HexA-GlcNAc(6S) ({Delta}Di-6S), {Delta}HexA-GlcN(NS,6S) ({Delta}Di-diS1), {Delta}HexA(2S)-GlcN(NS) ({Delta}Di-diS2), and {Delta}HexA(2S)-GlcN(NS,6S) ({Delta}Di-triS1).

 


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FIG. 3.
HPLC analysis on a gel filtration column of oligosaccharides derived from 35S-labeled heparan sulfate. Standard oligosaccharides were prepared from heparin as described in "Experimental Procedures." Then, 5 µg of each oligosaccharide, hexasaccharide (Hexa), tetrasaccharide (Tetra), and disaccharide (Di), was applied to a SuperdexTM Peptide HR10/30 column (panel A). The conditions for HPLC were as described under "Experimental Procedures." Panels B and C shows analysis of radioactive fractions obtained from the BioGel P-4 column, P4-1 and P4-2 in Fig. 2, respectively.

 

Structural Analysis of 35S-Labeled Disaccharides from Heparan Sulfate—The 35S-labeled disaccharide fraction (P4-2) was digested with 2-sufatase as described under "Experimental Procedures," and analyzed by HPLC on a SAX column. Fig. 4A shows the analysis of P4-2. With the enzyme digestion (Fig. 4B), only one component, peak 2 in panel A, shifted its retention time to an earlier position. The result indicates that peak 2 has a {Delta}HexA(2S) residue, but not peak 1. To confirm the location of sulfate group of glucosamine residues, P4-2 was treated with mercuric acetate to remove unsaturated uronic acids. The reaction products were reduced with sodium borohydride and analyzed by HPLC on a SAX column (Fig. 5A). By comparison with 3H-labeled standards (Fig. 5B), peak a and peak b were identified as reduced forms of GlcN(NS,3S) and GlcN-(NS,3S,6S). Judging from peak area, peak a and peak b originated from peak 2 and peak 1 in Fig. 4A, respectively. From these data, peak 1 and peak 2 in Fig. 4A were determined as {Delta}HexA-GlcN(NS,3S,6S) and {Delta}HexA(2S)-GlcN(NS,3S), respectively. Fig. 9, A and B show schematic drawings of the analysis described above.



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FIG. 4.
HPLC analysis of 35S-labeled disaccharides digested with 2-sulfatase. 35S-Labeled disaccharides derived from 3-OST-5-modified heparan sulfate (P4-2 in Fig. 2) were digested with 2-sulfatase as described under "Experimental Procedures." The digested products were analyzed by HPLC on a SAX column (panel B). Panel A shows analysis of untreated P4-2. A dotted line indicates the shifted peak.

 


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FIG. 5.
HPLC analysis of 35S-labeled disaccharides treated with mercuric acetate. 35S-Labeled disaccharides derived from 3-OST-5-modified heparan sulfate (P4-2 in Fig. 2) were treated with mercuric acetate to remove unsaturated uronic acid residues as described under "Experimental Procedures." The reaction products were reduced with sodium borohydride and analyzed by HPLC on a SAX column (panel A). Panel B shows analysis of sulfated glucosamine standards, GlcN(NS,3S) and GlcN(NS,3S,6S), reduced with sodium [3H]borohydride. A letter R attached to the name means reduced form.

 


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FIG. 9.
Schematic drawing of analysis. The method for the determination of three 3-O-sulfated disaccharides is outlined. {Delta}HexA-GlcN(NS,3S,6S) (A) and {Delta}HexA(2S)-GlcN(NS,3S) (B) were derived from 3-OST-5-modified heparan sulfate, and {Delta}HexA(2S)-GlcN(NS,3S,6S) (C) from 3-OST-5-modified heparin. Figure and peak numbers under each substance indicate where these substances were identified as a HPLC peak.

 

Highly Sulfated Structure of 35S-Labeled Heparin—35S-Labeled heparin was prepared by incubating heparin with [35S]PAPS and the recombinant 3-OST-5 and digested with a mixture of heparin lyases as described under "Experimental Procedures." When the digested products were separated by HPLC on a SAX column, major radioactivity was detected at the retention time of 30.1 min, later than the positions of trisulfated disaccharides (Fig. 6). The fraction indicated by a white arrow in Fig. 6 was pooled and desalted for further analysis. The 35S-labeled product was confirmed to be a disaccharide by HPLC on a gel filtration column (data not shown). On 2-sulfatase digestion, the 35S radioactive peak exhibited a shift in retention time to an earlier position (Fig. 7), indicative of a {Delta}HexA(2S) residue. The retention time of the 2-sulfatase-treated product corresponded to that of a disaccharide isolated from 3-OST-5-modified heparan sulfate (peak 1 in Fig. 4A), its structure being determined as {Delta}HexA-GlcN(NS,3S,6S) as described above. Therefore, {Delta}HexA(2S)-GlcN(NS,3S,6S) was expected. To confirm the location of sulfate group of glucosamine residue, 35S-labeled disaccharide was treated with mercuric acetate, reduced with sodium borohydride, and analyzed by HPLC on a SAX column (Fig. 8A). The position of the 35S radioactive peak corresponded to that of 3H-labeled GlcN (NS,3S,6S) standard (Fig. 8B). From these data, the 35S-labeled disaccharide from 3-OST-5-modified heparin was determined as {Delta}HexA(2S)-GlcN(NS,3S,6S). Fig. 9C shows a schematic drawing of the analysis described above.



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FIG. 7.
HPLC analysis of 35S-labeled disaccharide digested with 2-sulfatase. 35S-Labeled disaccharide derived from 3-OST-5-modified heparin (white arrow in Fig. 6) was digested with 2-sulfatase as described under "Experimental Procedures." The digested product was analyzed by HPLC on a SAX column (panel B). Panel A shows analysis of untreated 35S-labeled disaccharide. A dashed line indicates the shifted peak.

 


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FIG. 8.
HPLC analysis of 35S-labeled disaccharide treated with mercuric acetate. 35S-Labeled disaccharide derived from 3-OST-5-modified heparin (white arrow in Fig. 6) was treated with mercuric acetate to remove unsaturated uronic acid residue as described under "Experimental Procedures." The reaction product was reduced with sodium borohydride and analyzed by HPLC on a SAX column (panel A). Panel B shows analysis of sulfated glucosamine standards, GlcN(NS,3S) and GlcN(NS,3S,6S), reduced with sodium [3H]borohydride. A letter R attached to the name means reduced form.

 

Quantitative Analysis of the 3-OST-5 Transcripts in Human Tissues—We determined the tissue distribution and expression levels of the 3-OST-5 transcripts by a real-time PCR method. The expression levels of 3-OST-5 in various tissues were shown as a relative amount compared with the GAPDH transcripts (Fig. 10). The transcripts were highly expressed in fetal brain, followed by adult brain and spinal cord. Cerebellum, colon and skeletal muscle expressed the 3-OST-5 transcripts at a relatively low level. The expression levels in the remaining tissues were very low or undetectable.



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FIG. 10.
Quantitative analysis of 3-OST-5 transcripts in human tissues by real-time PCR. Standard curves for 3-OST-5 and GAPDH were generated by serial dilution of control DNA or plasmid DNA as described under "Experimental Procedures." The expression level of 3-OST-5 was normalized to that of the GAPDH transcript, which was measured in the same cDNAs. Data were obtained from triplicate experiments and are given as the mean ± S.D. PBMC, peripheral blood mononuclear cell.

 

Identification of Tetrasulfated Disaccharide in Bovine Intestinal Heparan Sulfate—To confirm the presence of the natural tetrasulfated disaccharide unit in heparan sulfate, we chose bovine intestinal heparan sulfate as a natural source because of the availability of highly purified material. The bovine intestinal heparan sulfate was digested with a mixture of heparin lyases as described under "Experimental Procedures." The digested products were reduced with sodium [3H]borohydride and subjected to HPLC on a SAX column (Fig. 11). Six major 3H radioactive peaks were identified as known disaccharides by comparing with disaccharide standards reduced with sodium borohydride. 35S-Labeled tetrasulfated disaccharide obtained from 3-OST-5-modified heparin as described above was reduced with sodium borohydride and used as a standard. Its elution position is shown in Fig. 11 as {Delta}Di-tetraSR. Weak but apparent radioactivity was detected at this position (inset of Fig. 11). To confirm the molecular size, the peak fraction (indicated by a white arrow) was analyzed by the HPLC on a gel filtration column. The 3H-labeled substance was eluted at the position of the tetrasulfated disaccharide standard (data not shown).



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FIG. 11.
HPLC analysis on a SAX column of 3H-labeled disaccharides derived from bovine intestinal heparan sulfate. Heparan sulfate from bovine intestine was digested with a mixture of heparin lyases as described under "Experimental Procedures." The digested products were reduced with sodium [3H]borohydride and separated by HPLC on a SAX column. The conditions for HPLC were as described under "Experimental Procedures." Fractions of 0.24 ml were collected, and radioactivity was determined. Six known disaccharides were identified in comparison with the disaccharide standards reduced with sodium borohydride. Abbreviations of disaccharides are shown in the legend of Fig. 1. A letter R attached to the name means reduced form. 35S-Labeled {Delta}HexA(2S)-GlcN(NS,3S,6S) obtained from 3-OST-5-modified heparin as described in the present study was reduced with sodium borohydride and used as a standard. Its elution position is indicated by {Delta}Di-tetraSR. sodium [3H]borohydride was eluted at the retention time of 7 min. The inset represents an enlarged vertical axis with the same horizontal scale. The fraction indicated by a white arrow was used for further analysis.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cloning and characterization of a sixth member of the human 3-OST family, named 3-OST-5, was recently reported. Xia et al. (35) employed low pH nitrous acid degradation to analyze the reaction products and identified three 3-O-sulfated disaccharides, GlcA-aMan(3S,6S), IdoA(2S)-aMan(3S), and IdoA(2S)-aMan(3S,6S). In the case of 3-O-sulfated glucosamine, both N-sulfated and N-unsubstituted residues are susceptible to low pH nitrous acid degradation, therefore the N-substituents of the glucosamine residue was unidentified (31). They also confirmed that the 3-OST-5-modified heparan sulfate binds to AT III and gD. In addition, transfection of the plasmid expressing 3-OST-5 makes CHO cells susceptible to HSV-1. From these results, they concluded that 3-OST-5 has the activities of both 3-OST-1 and 3-OST-3. The reaction specificity of 3-OST-1 and 3-OST-3 is the formation of GlcA-GlcN-(NS,3S,±6S) and Ido(2S)-GlcN(3S,±6S), respectively (31, 44).

In the present study, we employed enzyme digestion to analyze the reaction products. 35S-Labeled heparan sulfate, which was prepared by incubating heparan sulfate with [35S]PAPS and recombinant 3-OST-5, was digested with a mixture of heparin lyases. Two major 35S-labeled disaccharides obtained from the digests were determined as {Delta}HexA-GlcN(NS,3S,6S) and {Delta}HexA(2S)-GlcN(NS,3S). These two disaccharides appear to resemble the reaction products of 3-OST-1 and 3-OST-2, respectively, although the types of uronic acids are unidentified.

It has been believed that the glucosaminidic linkage adjacent to a disaccharide unit containing a 3-O-sulfated glucosamine residue is resistant to heparin lyases (45, 46). To date, unsaturated disaccharides containing a 3-O-sulfated glucosamine residue have not been isolated. On the other hand, Rhomberg et al. (47) reported that a decasaccharide containing a 3-O-sulfated glucosamine residue was depolymerized by heparinase II, and confirmed that the reaction product contained an unsaturated 3-O-sulfated disaccharide unit in the non-reducing terminal. They also examined a shorter oligosaccharide resistant to heparinase II and concluded that the length of the oligosaccharide determines the susceptibility to the enzyme. Accordingly, it is considered that the resistance of tetrasaccharides isolated in this study to the enzyme digestion may be due to the short length of the chain. Interestingly, the enzyme digestion of 3-OST-3-modified heparan sulfate produces no disaccharide containing a 3-O-sulfated glucosamine residue (31). At this time, the reaction specificity of heparin lyases especially for the 3-O-sulfated structure is still obscure. A more detailed analysis of the sulfated monosaccharide sequence around the modification site of 3-OST-5 will be helpful for solving this problem.

In addition to heparan sulfate, we analyzed 3-OST-5-modified heparin. HPLC analysis of 35S-labeled heparin digested with a mixture of heparin lyases showed a very different elution profile compared with heparan sulfate. A prominent peak was observed at the later position of trisulfated disaccharides, and its structure was determined as {Delta}HexA(2S)-GlcN (NS,3S,6S). As far as we know, this is the first report to show the enzyme catalyzes the reaction to form the tetrasulfated disaccharide unit. A small peak detected at the elution position of tetrasulfated disaccharide in Fig. 1B (peak 5) indicates enzymatic formation of this structure with heparan sulfate as an acceptor substrate.

3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord. Some other tissues, including skeletal muscle, also expressed 3-OST-5 at a relatively low level. Xia et al. (35) did not examine fetal brain in their Northern blot analysis. They also did not detect the expression of 3-OST-5 in the brain, and concluded that 3-OST-5 is predominantly expressed in skeletal muscle. This discrepancy is unexplained, although real time-PCR is a more quantitative method than Northern blot analysis. 3-OST-2 and 3-OST-4 were also predominantly expressed in human brain. 3-OST-1 and 3-OST-3 were widely expressed in human tissues (34). Heparan sulfate in the nervous system is predominantly expressed on proteoglycans of either the syndecan family (48) or the glypican family (49). A number of investigations have revealed that the development of the central nervous system is controlled by the spatiotemporal expression of these proteoglycans (50). Therefore, it can be expected that the 3-OSTs specifically expressed in the central nervous system play an important role in forming specific domain structures of heparan sulfate to bind some functional proteins necessary to control the central nervous system.

In the present study, we demonstrated the enzymatic formation of a tetrasulfated disaccharide unit in vitro. No evidence of this novel structure in natural heparan sulfate or heparin has been reported as far as we know. Therefore, we focused on the detection of the tetrasulfated disaccharide unit in natural heparan sulfate. We chose bovine intestinal heparan sulfate as a natural source because of the availability of highly purified material, even though the expression level of 3-OST-5 is relatively low in human intestine. As the result of analysis, bovine intestinal heparan sulfate was found to contain about 0.15% tetrasulfated disaccharide (mol/mol of total disaccharides). The retention time of tetrasulfated disaccharide on a SAX-column shifted markedly to an early position on reduction with sodium borohydride, although the retention times of six major disaccharides were not changed significantly by the reduction. Interestingly, the same phenomenon was observed with {Delta}HexA-GlcN(NS,3S,6S) and {Delta}HexA(2S)-GlcN(NS,3S). Therefore this is a characteristic of disaccharides containing a 3-O-sulfated glucosamine residue.

Although 3-OST-5 is the only enzyme known to catalyze the formation of the tetrasulfated disaccharide unit at this time, there is no evidence that the novel structure detected in this study was synthesized by the 3-OST-5 in bovine intestine. The generation of transgenic and gene knockout animal models could provide not only the answer to this question but also important information about the biological role of 3-OST-5 or its reaction products including tetrasulfated disaccharide.


    FOOTNOTES
 
* This work was supported by the R&D Project of the Industrial Science and Technology Frontier Program (R&D for Establishment and Utilization of a Technical Infrastructure for Japanese Industry) supported by the New Energy and Industrial Technology development Organization (NEDO). 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. Back

|||| To whom correspondence should be addressed: Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Tel.: 81-298-61-3200; Fax: 81-298-61-3201; E-mail: h.narimatsu{at}aist.go.jp.

1 The abbreviations used are: HSPG, heparan sulfate proteoglycan; AT III, antithrombin III; gD, glycoprotein D; 3-OST, heparan sulfate D-glucosaminyl 3-O-sulfotransferase; HSV-1, herpes simplex virus type-1; GlcA, D-glucuronic acid; IdoA, L-iduronic acid; GlcN, D-glucosamine; GlcNAc, N-acetyl-D-glucosamine; aMan, 2,5-anhydro-D-mannitol; {Delta}HexA, 4,5-unsaturated uronic acid; 2S, 2-O-sulfate; NS, 2-N-sulfate; 3S, 3-O-sulfate; 6S, 6-O-sulfate; {Delta}Di-0S, {Delta}HexA-GlcNAc; {Delta}Di-NS, {Delta}HexA-GlcN(NS); {Delta}Di-6S, {Delta}HexA-GlcNAc(6S); {Delta}Di-diS1, {Delta}HexA-GlcN(NS,6S); {Delta}Di-diS2, {Delta}HexA(2S)-GlcN(NS); {Delta}Di-triS1, {Delta}HexA(2S)-GlcN(NS,6S); {Delta}Di-triS2, {Delta}HexA-GlcN(NS,3S,6S); {Delta}Di-triS3, {Delta}HexA(2S)-GlcN(NS,3S); {Delta}Di-tetraS, {Delta}HexA(2S)-GlcN (NS,3S,6S); PAPS, 3'-phosphoadenosine 5'-phosphosulfate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SAX, strong anion-exchange; HPLC, high performance liquid chromatography. Back


    ACKNOWLEDGMENTS
 
We thank Hiroshi Kikuchi for excellent technical assistance, Dr. Mamoru Kyogashima for helpful support, and Dr. Hiroko Habuchi for critical reading of the manuscript.



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