Glycobiology Advance Access originally published online on September 23, 2007
Glycobiology 2007 17(12):1365-1376; doi:10.1093/glycob/cwm103
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Molecular Cloning of Squid N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase and Synthesis of a Unique Chondroitin Sulfate Containing E–D Hybrid Tetrasaccharide Structure by the Recombinant Enzyme
2 Department of Chemistry, Aichi University of Education, Igaya-cho, Kariya, Aichi 448-8542, Japan
3 Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
1 To whom correspondence should be addressed: Fax: +81-566-26-2649; e-mail: ohabuchi{at}auecc.aichi-edu.ac.jp
Received on June 9, 2007; revised on August 23, 2007; accepted on September 20, 2007
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Abstract |
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N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues in chondroitin sulfate (CS). We previously purified squid GalNAc4S-6ST and cloned a cDNA encoding the partial sequence of squid GalNAc4S-6ST. In this paper, we cloned squid GalNAc4S-6ST cDNA containing a full open reading frame and characterized the recombinant squid GalNAc4S-6ST. The cDNA predicts a Type II transmembrane protein composed of 425 amino acid residues. The recombinant squid GalNAc4S-6ST transferred sulfate preferentially to the internal GalNAc(4SO4) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO4) could be sulfated efficiently when the GalNAc(4SO4) residue was included in the unique nonreducing terminal structure, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), which was previously found in CS-A. Shark cartilage chondroitin sulfate C (CS-C) and chondroitin sulfate D (CS-D), poor acceptors for human GalNAc4S-6ST, served as the good acceptors for the recombinant squid GalNAc4S-6ST. Analysis of the sulfated products formed from CS-C and CS-D revealed that GalNAc(4SO4) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), were sulfated efficiently by squid GalNAc4S-6ST, and the E–D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4) was generated in the resulting sulfated glycosaminoglycans. These observations indicate that the recombinant squid GalNAc4S-6ST is a useful enzyme for preparing a unique chondroitin sulfate containing the E–D hybrid tetrasaccharide structure.
Key words: chondroitin sulfate D / chondroitin sulfate E / GalNAc4S-6ST / squid / sulfotransferase
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementFundingReferences |
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Chondroitin sulfate (CS) chains contain various repeating disaccharide structures. Chondroitin sulfate E (CS-E) and chondroitin sulfate D (CS-D) are characterized by unique repeating disaccharide units; E-disaccharide unit, GlcAß1-3GalNAc(4,6-SO4) ß1-4, for CS-E and D-disaccharide unit, GlcA(2SO4) ß1-3GalNAc(6SO4) ß1-4, for CS-D. CS-E has been reported to be involved in various physiological processes, such as immunological response of mast cells (Razin et al. 1982
; Stevens et al. 1983; Eliakim et al. 1986; Katz et al. 1986; Stevens et al. 1988; Davidson et al. 1990), regulation of procoagulant activity (McGee et al. 1995), promotion of neurite outgrowth (Clement et al. 1999; Tully et al. 2004), neural cell adhesion through binding to midkine (Ueoka et al. 2000), and enhancement of plasminogen activation by plasminogen activator (Sakai et al. 2000). CS-E was reported to inhibit the binding of versican to L-selectin or chemokines, and to bind L-selectin, chemokines (Kawashima et al. 2000; Hirose et al. 2001), and various heparin binding growth factors (Deepa et al. 2002). Cell surface CS-E was reported to participate in the infection of herpes symplex virus (Bergefall et al. 2005; Uyama et al. 2006). Intraperitoneally administered chondroitin sulfate E inhibited the development of antibody-induced arthritis (Yamamoto et al. 2006.). On the other hand, CS-D was reported to be involved in outgrowth of neurite (Clement et al. 1998) and binding of pleiotrophin (Maeda et al. 2003). D-unit-rich CSs were suggested to be involved in delimiting the border of the optic tract and in the chronological sorting of the retinal axons in chick embryo (Ichijo H. 2006).
The final steps in the syntheses of E-disaccharide unit and D-disaccharide unit are catalyzed by GalNAc4S-6ST and uronosyl 2-O-sulfotransferase (2OST), respectively. GalNAc4S-6ST transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of GalNAc(4SO4) residue (Habuchi et al. 1971; Ito and Habuchi 2000; Ohtake et al. 2001) and is a key enzyme for the synthesis of GalNAc (4,6-SO4) residues. On the other hand, 2OST transfers sulfate to position 2 of GlcA residue located in a unique sequence in CS, GalNAc(4SO4)-GlcA-GalNAc(6SO4), and position 2 of L-iduronic acid (IdoA) residue in IdoA-GalNAc(4SO4) and IdoA-GalNAc unit in dermatan sulfate (DS) and desulfated DS, respectively (Kobayashi et al. 1999; Ohtake et al. 2005). GalNAc4S-6ST was purified to the homogeneity from the squid cartilage (Ito and Habuchi 2000). On the basis of the amino acid sequence of the squid GalNAc4S-6ST, a partial cDNA of squid GalNAc4S-6ST was cloned (Ohtake et al. 2005). From the amino acid sequence deduced from the squid cDNA, we identified human GalNAc4S-6ST cDNA (Ohtake et al. 2005). Human GalNAc4S-6ST exhibited high activity toward the nonreducing terminal GalNAc(4SO4) residue of chondroitin sulfate A (CS-A) and was suggested to be involved in the unique nonreducing terminal modification of CS together with 2OST (Ohtake et al. 2003), whereas the purified squid GalNAc4S-6ST transferred sulfate mainly to the internal GalNAc(4SO4) residue of CS-A (Ito and Habuchi 2000). Such difference in the substrate recognition between human and squid GalNAc4S-6ST suggests that squid GalNAc4S-6ST may be a useful enzyme for in vitro enzymatic synthesis of CS-E. We have actually shown previously that the purified squid GalNAc4S-6ST could generate highly sulfated CS-E from CS-A (Habuchi et al. 2002). In this paper, we cloned full sized cDNA of squid GalNAc4S-6ST and characterized the recombinant enzyme. We found that the recombinant squid GalNAc4S-6ST could synthesize a unique chondroitin sulfate containing E–D hybrid tetrasaccharide structure.
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Results |
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Molecular cloning of squid GalNAc4S-6ST
Strategy for molecular cloning of cDNA containing a full open reading frame of squid GalNAc4S-6ST is shown in Figure 1A. Nucleotide sequence of cDNA-1 and amino acid sequence of peptide 1 to 5 were reported previously (Ohtake et al. 2001
). Sequences of oligonucleotides used are listed in Table I. The nucleotide sequences of the squid GalNAc4S-6ST cDNA, and the predicted amino acid sequences are shown in Figure 2. This cDNA predicts a protein composed of 425 amino acids. A putative hydrophobic transmembrane domain was found in the amino-terminal region, 21 residues in length, which covers amino acid residues 7–27. All the amino acid sequences of peptide 1 to 5 that had been obtained from the purified protein were found in the predicted protein sequences. Putative PAPS binding sites, KSGT (5'-PSB) and RNPADRLFS (3'-PB), (Kakuta et al. 1997) were present in the amino acid residue, 141–144 and 256–264, respectively. There are seven potential N-glycosylation sites in squid GalNAc4S-6ST, coinciding with the previous observation that the purified squid GalNAc4S-6ST contained abundant N-glycans. A tentative polyadenylation signal was found in the coding region (nucleotide number 1573–1578).
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Comparison of the coding sequence of squid GalNAc4S-6ST with that of the human counterpart revealed that there is 39% identity between squid GalNAc4S-6ST and human GalNAc4S-6ST at the amino acid level (Figure 3). The most prominent difference in the amino acid sequences between squid and human GalNAc4S-6ST is deletion of 95 amino acid residues at the N-terminal region of the squid protein. Additional deletion of amino acid residues were observed at the amino acid residues, 34, 58, 85, 180, and 200. Human GalNAc4S-6ST contains five potential N-glycosylation sites (Ohtake et al. 2001
). Of the seven potential N-linked glycosylation sites present in squid GalNAc4S-6ST, only one N-linked glycosylation site located behind 3'-PB (Asn273 indicated by a dot in Figure 3) was conserved between squid and human GalNAc4S-6ST.
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Substrate specificity of the recombinant squid GalNAc4S-6ST
To determine the substrate specificity of the expressed enzyme, we prepared the affinity-purified protein from the extracts of COS-7 cells transfected with pFLAGsGalNAc4S-6ST. The recombinant protein was prepared as described in Materials and methods, and was visualized with Western blot before or after N-Glycosidase F digestion (Figure 1B). After N-Glycosidase F digestion, a single protein band was detected at the migration position of 52 kDa that agreed well with the molecular weight, 52319, calculated from the cDNA. A weak band is visible in the Western blot at 155 kDa before N-Glycosidase F digestion and was shifted to the position of 105 kDa after N-Glycosidase F digestion. These peaks may be dimers, but were not examined further. In Table II, the sulfotransferase activities toward various glycosaminoglycans and oligosaccharides are shown. Squid GalNAc4S-6ST could sulfate CS-A, chondroitin sulfate C (CS-C), DS and various oligosaccharides containing GalNAc(4SO4) residue. The rate of sulfation of CS-C and CS-D by squid GalNAc4S-6ST was 28% and 55%, respectively, of that of CS-A. The relatively high activity toward CS-C and CS-D is contrasted with the activity of human GalNAc4S-6ST; the rate of sulfation of CS-C by human GalNAc4S-6ST was less than 2% of that of CS-A (Ohtake et al. 2001
). It is not examined whether the low sulfotransferase activity toward heparan sulfate and CDSNS–heparin is due to the contaminating glycosaminoglycans such as DS. Oligosaccharides used in this study are listed in Table III. Among trisaccharides used as acceptors, Oligo I and Tri-44 were the best acceptors. Tri-44 was a better acceptor than Tri-64, and Tri-64 was a better acceptor than Tri-46. 
Tetra AD served as the acceptor, but the rate of sulfation of Tetra AD was lower than that of Oligo I, suggesting that nonreducing terminal unsaturated uronate may inhibit the enzyme activity. The nonsulfated trisaccharide did not serve as the acceptor at all.
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Structural analysis of the 35S-labeled glycosaminoglycans
To determine the position to which 35SO4 was transferred from [35S]PAPS, we digested 35S-labeled CS-A with chondroitinase ACII, and analyzed by Partisil-10 SAX high performance liquid chromatography (HPLC) (Figure 4A–C). The radioactivity was detected at the position of
Di-diSE and Oligo II, and only trace amount of radioactivity was observed at the position of GalNAc (4, 6-SO4) (Figure 4A). After chondro-6-sulfatase digestion, the radioactivity was shifted to the position of inorganic sulfate and to the position slightly behind Di-diSE (Figure 4B). We have shown previously that chondro-6-sulfatase removes 6-O-sulfate group from not only Di-diSE, but also GalNAc(4,6-SO4) (Ito and Habuchi 2000), and 6-O-sulfate group located at the reducing terminal GalNAc residues of trisaccharides. The latter peak observed in Figure 4B was shown to be an oligosaccharide derived from Oligo II by desulfation of the reducing terminal GalNAc(6SO4) residue by chodro-6-sulfatase (Ohtake et al. 2003). These separation patterns are much different from those obtained from the analysis of 35S-labeled CS-A synthesized by human GalNAc4S-6ST; when 35S-labeled CS-A synthesized by human GalNAc4S-6ST was digested with chondroitinase ACII, major radioactivity was detected at the position of GalNAc(4,6-SO4) in addition to Di-diSE and Oligo II (Ohtake et al. 2003). When 35S-glycosaminoglycans formed from sturgeon notochord CS-A by the sulfation with squid GalNAc4S-6ST was digested with chondroitinase ACII and analyzed by SAX-HPLC, most of the radioactivity was detected at the position of Di-diSE and less than 5% of the radioactivity was detected at the position of GalNAc(4,6-SO4) (Figure 4C). We have previously found that, when sturgeon notochord CS-A was sulfated by human GalNAc4S-6ST and the sulfated product was analyzed by SAX-HPLC after chondroitinase ACII digestion, radioactivity was detected at the position of GalNAc(4,6-SO4) and Di-diSE in a nearly equal ratio, but not at all at the position of Oligo II (Ohtake et al. 2003), suggesting that sturgeon notochord CS-A may be devoid of the unique nonreducing terminal structure, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), that is to undergo nonreducing terminal modification by GalNAc4S-6ST. Taken together, it is strongly suggested that squid GalNAc4S-6ST transfers sulfate mainly to the internal GalNAc(4SO4) residues, and that sulfation of the nonreducing terminal GalNAc(4SO4) residue by squid GalNAc4S-6ST hardly occurs unless the GalNAc(4SO4) residue is included in the unique sequence, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4).
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Unlike human GalNAc4S-6ST, squid GalNAc4S-6ST could sulfate CS-C and CS-D efficiently. When 35S-labeled glycosaminoglycans formed from CS-C and CS-D were digested with chondroitinase ACII and separated with SAX-HPLC, two radioactive peaks were observed (Figure 4D and F); one was eluted at the position of
Di-diSE and another was eluted later than Di-triS. When the 35S-labeled glycosaminoglycans formed from CS-C and CS-D were digested with chondroitinase ABC, only Di-diSE was detected (Figure 4E and G). These observations suggest that the unknown peak contains an oligosaccharide resistant to chondroitinase ACII; therefore, the material included in this peak was designated as Oligo X. Because GlcA(2SO4)-containing sequences in CS exhibited various degree of resistance to chondroitinase ACII (Ohtake et al. 2003), and both CS-C and CS-D from shark cartilage are known to contain GlcA(2SO4)-GalNAc(6SO4) unit (D-disaccharide unit), it is possible that Oligo X was excised from a tetrasaccharide sequence containing GlcA-GalNAc(4,6-SO4) (E-disaccharide unit) and D-disaccharide unit. To confirm this possibility, Oligo X was purified with Superdex 30 chromatography and subjected to structural analysis. Oligo X was eluted at the same positions as those of Tetra ED in Superdex 30 chromatography (Figure 5A) and SAX-HPLC (Figure 5B). When Oligo X was digested with chondroitinase ACII under strong conditions and applied to SAX-HPLC, the radioactivity appeared at the position of Di-diSE (Figure 5C), and shifted to the position of inorganic sulfate after chondro-6-sulfatase digestion (Figure 5D). These results clearly indicate that the apparent resistance of Oligo X toward chondroitinase ACII is not due to the presence of iduronic acid residue. We have previously obtained Oligo II from the nonreducing end of CS-A after chondroitinase ACII digestion (Ohtake et al. 2003). If Oligo X is an unsaturated tetrasaccharide containing E-disaccharide and D-disaccharide, HexA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4), mercuric acetate treatment of Oligo X should yield Oligo II. To confirm this, Oligo X was treated with mercuric acetate and the resulting product was separated with SAX-HPLC. The mercuric acetate-treated Oligo X behaved identically with Oligo II in SAX-HPLC (Figure 5E). After digestion with chondroitinase ACII, the radioactivity was shifted to the position of GalNAc(4,6-SO4) (Figure 5F). Taken together, it is most probable that Oligo X is an unsaturated E–D hybrid tetrasaccharide, HexA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4).
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Structural analysis of the 35S-labeled oligosaccharides
To determine the position to which 35SO4 was transferred, the 35S-labeled oligosaccharides formed from Tri-46, Tri-64, and Tri-44 by squid GalNAc4S-6ST were digested with chondroitinase ACII, and applied to SAX-HPLC. Only GalNAc(4,6-SO4) was formed from the 35S-labeled Tri-46 (Figure 6A), whereas only
Di-diSE was obtained from the 35S-labeled Tri-64 (Figure 6B). Because the rate of sulfation of Tri-46 was 58% of the rate of sulfation of Tri-64 (Table II), squid GalNAc4S-6ST appears to transfer sulfate to the reducing terminal GalNAc(4SO4) more efficiently than the nonreducing terminal GalNAc(4SO4). Such property of squid GalNAc4S-6ST was also supported by the analysis of the 35S-labeled Tri-44; from the 35S-labeled Tri-44, 82.2% and 17.8% of the radioactivity was recovered in Di-diSE and GalNAc (4, 6-SO4), respectively (Figure 6C). In contrast, when Tri-44 was sulfated by human GalNAc4S-6ST, only GalNAc (4, 6-SO4) was formed after chondroitinase ACII digestion (Ohtake et al. 2001). When the 35S-labeled Tetra AD was digested with chondroitinase ACII under strong conditions and subjected to SAX-HPLC, radioactivity was detected only at Di-diSE (Figure 6D), indicating that the sulfated product is Tetra ED. The Km and the Vmax for Tri-46, Tri-44, Tri-64, and Oligo I were compared (Table IV). The affinity deduced from the Km values was the lowest for Tri-46 and the highest for Oligo I, suggesting that the sulfation of position 6 of GalNAc(4SO4) residue at the nonreducing end by squid GalNAc4S-6ST was stimulated by the 2-O-sulfation of the penultimate GlcA residue as observed in human GalNAc4S-6ST.
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Enzymatic synthesis of a chondroitin sulfate containing E–D hybrid tetrasaccharide structure
To confirm that D-disaccharide unit is present in Oligo X, we tried to prepare nonradioactive Oligo X. CS-C was incubated with squid GalNAc4S-6ST in the presence of 2 mM PAPS as described under "Materials and methods". When the resulting reaction products formed from CS-C was digested with chondroitinase ABC and subjected to SAX-HPLC, the peak of
Di-diSE, which was less than 1% of total disaccharide units in intact CS-C (Figure 7A) became 15% (Figure 7B) of the total disaccharide units. When intact CS-C was subjected to a limited digestion with chondroitinase ACII followed by SAX-HPLC, a peak corresponding to Tetra AD was observed (Figure 7C). In contrast, when the sulfated CS-C was digested with chondroitinase ACII under the same limited conditions, the peak corresponding to Tetra AD was markedly decreased and two new peaks appeared at the position of Oligo X and Di-diSE (Figure 7D). These observations indicate that sulfation of CS-C with squid GalNAc4S-6ST in the presence of 2 mM PAPS resulted in a conversion of the A–D tetrasaccharide sequence from which Tetra AD was excised to the sequence from which Oligo X was formed. When Oligo X thus obtained was digested with chondroitinase ACII under strong conditions and separated by SAX-HPLC, two peaks corresponding to Di-diSE and Di-diSD were obtained (Figure 7F), confirming that Oligo X contains D-disaccharide unit. The structural analysis of Oligo X is summarized in Scheme 1. From the analytical data of Oligo X, we concluded that Oligo X is the unsaturated E–D hybrid tetrasaccharide, HexA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4), identical to Tetra ED. These observations clearly indicate that squid GalNAc4S-6ST catalyzes production of a unique chondroitin sulfate containing E–D hybrid tetrasaccharide structure.
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Discussion |
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In this paper, we cloned squid GalNAc4S-6ST cDNA. As far as we know, this is the first report for the cloning of molluscan sulfotransferase involved in the synthesis of glycosaminoglycans. Most remarkable difference between squid and human GalNAc4S-6ST is deletion of 95 amino acid residues at the N-terminal region of the squid protein. Nevertheless, these proteins appear to be well conserved during evolution because a significant homology (39% identity) is present between the enzyme obtained from the two species. Of the seven potential N-glycosylation sites present in squid GalNAc4S-6ST, only one N-linked glycosylation site was conserved between squid and human GalNAc4S-6ST. It remains to be studied whether the N-glycan conserved between squid and human GalNAc4S-6ST might affect the activity as observed in chondroitin 4-sulfotransferase and chondroitin 6-sulfotransferase (Yusa et al. 2005
, 2006).
Acceptor substrate specificity of the recombinant squid GalNAc4S-6ST is similar to that of human GalNAc4S-6ST; however, some significant differences are observed. Structural analysis of the 35S-labeled glycosaminoglycan formed from CS-A by squid GalNAc4S-6ST revealed that squid GalNAc4S-6ST transferred sulfate to position 6 of GalNAc(4SO4) residues located mainly to the internal region of CS-A, and sulfation of the nonreducing terminal GalNAc(4SO4) residue appears to occur when the GalNAc(4SO4) residue is located in the unique sequence, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6O4). The nonreducing terminal GalNAc(4SO4) residue that is not included in the unique sequence is thought to be a poor acceptor site for squid GalNAc4S-6ST, because the 35S-labeled glycosaminoglycan formed from sturgeon notochord CS-A by the sulfation with squid GalNAc4S-6ST yielded solely Di-diSE after digestion with chondroitinase ACII. In contrast, the 35S-labeled product formed from the same substrate by the sulfation with human GalNAc4S-6ST released both GalNAc(4, 6-SO4) and Di-diSE after chondroitinase ACII digestion (Ohtake et al. 2003). When sturgeon notochord CS-A was sulfated by either human or squid GalNAc4S-6ST, Oligo II was not obtained from the 35S-labeled products after digestion with chondroitinase ACII, suggesting that the unique nonreducing terminal sequence, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), might not be present in sturgeon notochord CS-A. We used affinity-purified FLAG-tagged enzyme for the characterization of the substrate specificity. At present, it is not clear whether the sulfotransferase activity and the substrate specificity may be affected by FLAG peptide.
A clear difference between squid and human GalNAc4S-6ST was observed when a trisaccharide Tri-44 was used as the acceptor; human GalNAc4S-6ST transferred sulfate exclusively to the nonreducing terminal GalNAc(4SO4) residue (Ohtake et al. 2001), whereas squid GalNAc4S-6ST transferred sulfated mainly to the reducing terminal GalNAc(4SO4) residue.
Squid GalNAc4S-6ST could sulfate CS-C and CS-D efficiently, whereas these glycosaminoglycans were relatively poor acceptors for human GalNAc4S-6ST (Ohtake et al. 2001). However, direct comparison between squid and human sulfotransferase activity is difficult because the assay conditions of these sulfotransferases are different and the efficiency of transfection of each experiment was not determined. When the sulfated products formed from CS-C and CS-D by squid GalNAc4S-6ST were digested with chondroitinase ACII, a novel oligosaccharide (Oligo X) was generated. Structural analysis of Oligo X revealed that Oligo X was an unsaturated tetrasaccharide, HexA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4). Oligo X should be derived from the E–D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4). Conversion of the precursor A-D tetrasaccharide sequence, GlcA-GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4), to the E–D hybrid tetrasaccharide sequence appeared to proceed nearly quantitatively by sulfation with squid GalNAc4S-6ST in the presence of 2 mM PAPS, because Tetra AD, which was released from intact CS-C by chondroitinase ACII digestion, was markedly decreased and a new peak corresponding to Tetra ED was released from the sulfated CS-C by chondroitinase ACII digestion. CS-D from shark cartilage was reported to contain GlcA(2SO4)-GalNAc(6SO4) unit (D-disaccharide unit) at the reducing side of GlcA-GalNAc(4SO4) unit (A-disaccharide unit) (Sugahara et al. 1996; Nadanaka and Sugahara 1997; Nadanaka et al. 1998). The nearly quantitative conversion of the A-D sequence to the E–D sequence by sulfation with squid GalNAc4S-6ST appears to be consistent with the reported sequence. Under the sulfation conditions, major part of GlcA-GalNAc(4SO4) units in CS-C still remained, suggesting that squid GalNAc4S-6ST may transfer sulfate preferentially to GalNAc(4SO4) residue adjacent to the nonreducing side of D-disaccharide unit. We have shown previously that whale cartilage CS-A contains the A-D sequence because Tetra AD was obtained after chondroitinase ACII digestion (Ohtake et al. 2005). When whale cartilage CS-A was sulfated by squid GalNAc4S-6ST and [35S]PAPS, about 9% of the radioactivity was detected at the position of Oligo X after chondroitinase ACII digestion. In contrast, when the same CS-A preparation was sulfated by human GalNAc4S-6ST and [35S]PAPS, no radioactivity was detected at the position of Oligo X after chondroitinase ACII digestion (data not shown). Because the content of D-disaccharide unit in whale cartilage CS-A was as much as 0.5% of the total disaccharide units, squid GalNAc4S-6ST may have much higher affinity for the A-D sequence than human GalNAc4S-6ST.
We showed that shark cartilage CS-D was a good acceptor for squid GalNAc4S-6ST. Sulfation of CS-D by squid GalNAc4S-6ST could provide a highly sulfated glycosaminoglycan containing E–D hybrid structure. As far as we know, such a sequence has not been found in the internal region of vertebrate chondroitin sulfate. It is of interest to determine whether the highly sulfated CS containing E–D hybrid structure would exhibit various biological activities observed in CS-E and CS-D.
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Materials and methods |
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Materials
The following commercial materials were used: H235SO4 was from Perkin-Elmer, Boston, MO, chondroitinase ACII, chondroitinase ABC, chondro-6-sulfatase, CS-A (whale cartilage), CS-C (shark cartilage), CS-D (shark cartilage), DS (pig skin), heparan sulfate (bovine liver), completely desulfated N-resulfated heparin (CDSNS-heparin),
Di-0S, Di-6S, Di-4S, Di-diSD, Di-diSE, and Di-triS were from Seikagaku Corporation, Tokyo; Partisil-10 SAX was from Whatman Japan, Tokyo, Japan, CS-A (sturgeon notochord), unlabeled PAPS, N-acetylgalactosamine 4-sulfate, N-acetylgalactosamine 6-sulfate were from Sigma, St. Louis, MO, N-Glycosidase F was from Roche Diagnostics, Tokyo, Japan, Hiload Superdex 30 HR 16/60 and Fast Desalting Column HR 10/10 were from GE Healthcare Biosciences, Piscataway, NJ.
[35S]PAPS was prepared as described (Delfert and Conrad 1985). Chondroitin (squid skin) was prepared as described (Habuchi and Miyata 1980). The FLAG-tagged human GalNAc4S-6ST was prepared as described previously (Ohtake et al. 2001). N-Acetylgalactosamine 4, 6-bissulfate was prepared from Di-diSE, as described previously (Ohtake et al. 2005). Keratan sulfate (bovine cornea) was a generous gift from Seikagaku Corporation. CS-E (squid cartilage), which was eluted with 1.5 M NaCl from DEAE-Sephadex A-50, was prepared as described (Habuchi et al. 1977). Desulfated DS was prepared from DS by the method of Nagasawa et al. (Nagasawa et al. 1979). Solvolysis with 90% (v/v) DMSO was performed at 100°C for 60 min. Disaccharide compositions of glycosaminoglycans were determined by absorbance at 232 nm of unsaturated disaccharides separated by SAX-HPLC after chondroitinase ACII or ABC digestion. Analytical data of these glycosaminoglycans are the same as those described in the previous paper (Ohtake et al. 2005) except for CS-D. The disaccharide composition of CS-D determined after chondroitinase ABC digestion was as follows: Di-0S 1.0%, Di-6S 36.4%, Di-4S 39.1%, Di-diSD 23.4%, and Di-diSE 0.1%.
The trisaccharides GalNAc(4SO4)-GlcA-GalNAc(4SO4) (Tri-44), GalNAc(4SO4)-GlcA-GalNAc(6SO4) (Tri-46), GalNAc(6SO4)-GlcA-GalNAc(4SO4) (Tri-64), GalNAc- (4SO4)-GlcA(2SO4)-GalNAc(6SO4) (Oligo I), and GalNAc- (4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4) (Oligo II) were prepared as described (Ito and Habuchi O. 2000; Ohtake et al. 2003, 2005). The unsaturated tetrasaccharide HexA-GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) (Tetra AD, which was called Tetra A in the previous paper) was prepared as described (Ohtake et al. 2005). [35S]HexA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4) (Tetra ED) was prepared from Tetra AD by the sulfation with squid GalNAc4S-6ST and [35S]PAPS as described below. It is evident that 35S-labeled oligosaccharide derived from Tetra AD is Tetra ED because radioactivity was detected in Di-diSE when the oligosaccharide was digested with chondroitinase ACII and subjected to SAX-HPLC (Figure 6D). Fresh squids, Ommastrephes sloani pacificus, were obtained locally.
Preparation of total RNA from the squid cartilage
Squid cranial cartilage was dissected, freed of soft tissues by wiping with cotton cloth and put into liquid nitrogen. The frozen cartilage was ground to powder in a mortar in the presence of liquid nitrogen. The cartilage powder was placed in 10 volumes of an ice-cold guanidine thiocyanate solution and homogenized with a Polytron homogenizer. The homogenate was centrifuged at 100,000 x g for 30 min. The clear supernatant fraction was used for isolation of total RNA by the guanidine thiocyanate/CsCl methods (Kingston 1996).
Construction of pFLAGsGalNAc4S-6ST and preparation of the affinity-purified protein
A cDNA encoding full open reading frame was amplified by PCR using cDNA-B as a template. The 5' and 3' primers were F5 and R11, respectively. At 5'-end of the oligonucleotide primers, restriction enzyme recognition sites were introduced; HindIII site for the sense primer F5 and EcoRI site for the antisense primer R11. The PCR product was digested with EcoRI and HindIII, and subcloned into these sites of pFLAG-CMV-2 plasmid (Kodak, New Haven, CT). The resulting plasmid, pFLAGsGalNAc4S-6ST, was transfected in COS-7 cells and the fusion protein produced was extracted with 0.15 M NaCl, 10 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 2 mM CaCl2, 0.5% Triton X-100, and 20% glycerol for 30 min on a rotary shaker. The extracts were centrifuged at 10,000 x g for 10 min. The sulfotransferase activities in the supernatant fractions were determined using CS-A as the acceptor. The GalNAc4S-6ST activity of the cells infected with the plasmid containing the squid cDNA was increased more than 7.5-fold over the activity of the cells infected with the vector alone (data not shown). The cellular extracts from ten 10-cm dishes were applied to an anti-FLAG mAb-conjugated agarose column (0.5 mL) (Sigma). The absorbed materials were eluted with 1.5 mL of a buffer containing FLAG peptide under the conditions recommended by the manufacturer.
Western blot analysis
The affinity-purified squid GalNAc4S-6ST was precipitated with two volumes of ethanol containing 1.3% (w/v) potassium acetate and digested with recombinant N-Glycosidase F (Roche Molecular Biochemicals) by the methods recommended by the manufacturer. After digestion, the samples were separated by SDS-polyacrylamide gel electrophoresis as described by Laemmli (Laemmli 1970). The separated proteins were electrophoretically transferred to an Immobilon-P membrane (Nihon Millipore, Tokyo, Japan), and stained with anti-FLAG M2 monoclonal antibody (Sigma). The blot was developed with polyclonal anti-mouse IgG antibody coupled to horse radish peroxidase using an ECL detection kit and a Hyperfilm ECL (Amersham Bioscience).
Assay of sulfotransferase activity
GalNAc4S-6ST activity was assayed by the method described previously (Ito and Habuchi O. 2000). The standard reaction mixture contained, in a final volume of 50 µL, 2.5 µmol of imidazole-HCl, pH 6.8, 1 µmol CaCl2, 1 µmol reduced glutathione, 25 nmol (as galactosamine) of CS-A or oligosaccharides, 50 pmol of [35S]PAPS (about 5.0 x 105 cpm), and enzyme. The enzymatic reaction was carried out at 20°C for 60 min because the optimum temperature was peaked at 20°C (data not shown). The reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. After the reaction was stopped, 35S-labeled glycosaminoglycans were isolated by the precipitation with ethanol followed by gel chromatography with a Fast Desalting Column as described previously (Habuchi et al. 1993), and the radioactivity was determined. When oligosaccharides were used as acceptors, the reaction mixtures were applied directly to the Superdex 30 column as described below, and the 35S -labeled oligosaccharides were separated from 35SO4 and [35S]PAPS. To obtain the kinetics parameters for oligosaccharides, the sulfotransferase activity was determined at the concentration of 5, 10, 20, 50, and 100 µM. The kinetics parameters were determined by a set of the Lineweaver-Burk's plot.
Enzymatic synthesis of glycosaminoglycans containing both E-disaccharide unit and D-disaccharide unit from CS-C
The affinity-purified squid GalNAc4S-6ST prepared as above was concentrated about 20-fold with Centricon YM-30 (Millipore Inc.). The reaction mixture was the same as that described above except that 25 nmol (as galactosamine) of CS-C, 100 nmol PAPS (final 2 mM) and 5 µL of the concentrated squid GalNAc4S-6ST were included. The enzymatic reaction was carried out at 15°C for 24 h. The reaction was stopped by immersing the reaction tubes in a boiling water bath for 1 min. After the reaction product was isolated by the precipitation with ethanol followed by gel chromatography with a Fast Desalting Column, the product was subjected to the second enzymatic reaction under the same conditions.
Digestion with chondroitinase ACII, chondroitinase ABC and chondro-6-sulfatase
Unless otherwise stated, digestion with chondroitinase ACII or chondroitinase ABC under the standard conditions was carried out for 4 h at 37°C in the reaction mixture containing, in a final volume of 25 µL, 1.25 µmol of Tris-acetate buffer, pH 7.5, 2.5 µg of bovine serum albumin and 30 mU of chondroitinase ACII or chondroitinase ABC. For degrading oligosaccharides containing GlcA(2SO4) residue with chondroitinase ACII, a strong condition was used under which digestion with chondroitinase ACII was carried out in the reaction mixtures described above three times successively; first with 120 mU enzyme for 28 h, second with 100 mU enzyme for 18 h, and finally with 100 mU enzyme for 7 h. The new enzymes were added after heating the reaction mixtures at 100°C for 1 min. To obtain the E–D hybrid tetrasaccharide from the sulfated CS-C, the sulfated products were subjected to a limited digestion with chondroitinase ACII; digestion was carried out for 1 h at 37°C in the reaction mixture described above except that 2 mU of chondroitinase ACII was included. After digestion of 35S-labeled glycosaminoglycans or oligosaccharides with chondroitinase ACII, digestion with chondro-6-sulfatase was carried out for 5 h at 37°C in the reaction mixtures containing, in a final volume of 25 µL, 1.25 µmol of Tris-acetate buffer, pH 7.5, 2.5 µg of bovine serum albumin and 100 mU of chondro-6-sulfatase, and repeated once more with the newly added enzyme for 24 h.
Removal of unsaturated uronic acid by mercuric acetate
Removal of unsaturated uronic acid was carried out as described (Ludwigs et al. 1987). Oligosaccharides containing unsaturated uronic acid were dried and dissolved in 1 mL of 35 mM mercuric acetate in 25 mM Tris/25 mM sodium acetate, pH 5.0. The reaction was carried out for 2 h at room temperature. After the reaction was over, the samples were applied to Dowex 50 (H+) column (bed volume of 1 mL). The column was washed with 3 mL of water. The flow through fractions and the washings were combined and lyophilized. The lyophilized materials were further purified with Superdex 30 and SAX-HPLC.
Chromatography on Superdex 30 and HPLC
A Superdex 30 16/60 column was equilibrated with 0.2 M NH4HCO3, and run at a flow rate of 2 mL/min. One milliliter fractions were collected. Separation of the degradation products formed from 35S-labeled glycosaminoglycans and 35S-labeled oligosaccharides were carried out by HPLC using a Whatman Partisil-10 SAX column (4.6 mm x 25 cm) equilibrated with 5 mM KH2PO4. The column was developed with a gradient (5 mM KH2PO4 for 10 min followed by a linear gradient from 5 to 500 or 720 mM KH2PO4); the gradient used was indicated in each figure. Fractions (0.5 mL) were collected at a flow rate of 1 mL/min and a column temperature of 40°C.
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Conflict of interest statement |
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None declared.
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Footnotes |
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The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession number AB292855.
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Funding |
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Ministry of Education, Culture, Sports, Science and Technology of Japan; Seikagaku Corporation.
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Abbreviations |
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A-disaccharide unit, GlcAß1-3GalNAc(4SO4) ß1-4; CS, chondroitin sulfate; CS-A, chondroitin sulfate A; CS-C, chondroitin sulfate C; CS-D, chondroitin sulfate D; CS-E, chondroitin sulfate E; D-disaccharide unit, GlcA(2SO4) ß1-3GalNAc(6SO4) ß1-4;
Di-0S, 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-D-galactose; Di-6S, 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; Di-4S, 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose; Di-diSD, 2-acetamide-2-deoxy-3-O-(2-O-sulfo-ß-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; Di-diSE, 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-4,6-bis-O-sulfo-D-galactose; Di-triS, 2-acetamide-2-deoxy-3-O-(2-O-sulfo-ß-D-gluco-4-enepyranosyluronic acid)-4,6-bis-O-sulfo-D-galactose; DS, dermatan sulfate; E-disaccharide, unit; GlcA, D-glucuronic acid; GlcAß1-3GalNAc(4,6-SO4), ß1-4; GalNAc4S-6ST, N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase; GlcA(2SO4), 2-O-sulfo-D-glucuronic acid; GalNAc(4SO4), 4-O-sulfo-N-acetylgalactosamine; GalNAc(6SO4), 6-O-sulfo-N-acetylgalactosamine; GalNAc(4,6-SO4), 4,6-bis-O-sulfo-N-acetylgalactosamine; HexA, 4,5-unsaturated hexuronic acid; HPLC, high performance liquid chromatography; IdoA, L-iduronic acid; Oligo I, GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4); Oligo II, GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4); 2OST, uronosyl 2-O-sulfotransferase; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; Tetra AD, HexA-GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4); Tetra ED, HexA-GalNAc(4,6-SO4)-GlcA(2SO4)-GalNAc(6SO4); Tri-44, GalNAc(4SO4)-GlcA-GalNAc(4SO4); Tri-46, GalNAc(4SO4)-GlcA-GalNAc(6SO4); Tri-64, GalNAc(6SO4)-GlcA-GalNAc(4SO4) |
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