Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO(4))) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO(4)) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO(4)) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.     

Molecular cloning and expression of chondroitin 4-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456-2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed approximately 29% identity and approximately 48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.     

Purification and characterization of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase from the squid cartilage

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate in chondroitin sulfate and dermatan sulfate, was purified 19,600-fold to apparent homogeneity from the squid cartilage. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a broad protein band with a molecular mass of 63 kDa. The protein band coeluted with GalNAc4S-6ST activity from Toyopearl HW-55 around the position of 66 kDa, indicating that the active form of GalNAc4S-6ST may be a monomer. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the internal N-acetylgalactosamine 4-sulfate residues. Chondroitin sulfate E, keratan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin were not efficient acceptors of the sulfotransferase. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine was used as acceptor, efficient sulfation of position 6 at the nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed.     

Molecular cloning and characterization of GalNAc 4-sulfotransferase expressed in human pituitary gland

We have previously cloned chondroitin-4-sulfotransferase (C4ST) cDNA from mouse brain. In this paper, we report cloning and characterization of GalNAc 4-sulfotransferase (GalNAc4ST), which transfers sulfate to position 4 of the nonreducing terminal GalNAc residue. The obtained cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 424 amino acid residues. Identity of the amino acid sequence between GalNAc4ST and human C4ST was 30%. When the cDNA was transfected in COS-7 cells, sulfotransferase activity toward carbonic anhydrase VI was overexpressed but no sulfotransferase activity toward chondroitin or desulfated dermatan sulfate was increased over the control. Sulfation of carbonic anhydrase VI by the recombinant GalNAc4ST occurred at position 4 of the GalNAc residue of N-linked oligosaccharides. The recombinant GalNAc4ST transferred sulfate to position 4 of GalNAc residue of p-nitrophenyl GalNAc, indicating that this sulfotransferase transfers sulfate to position 4 at the nonreducing terminal GalNAc residue. Dot blot analysis showed that the message of GalNAc4ST was expressed strongly in the human pituitary, suggesting that the cloned GalNAc4ST may be involved in the synthesis of the nonreducing terminal GalNAc 4-sulfate residues found in the N-linked oligosaccharides of pituitary glycoprotein hormones.     

Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis

4-O-Sulfation of GalNAc is a high frequency modification of chondroitin sulfate and dermatan sulfate (DS), and three major GalNAc 4-O-sulfotransferases including dermatan 4-O-sulfotransferase-1 (D4ST-1) and chondroitin 4-O-sulfotransferases-1 and -2 (C4ST-1 and -2) have been identified. 4-O-Sulfation of GalNAc during DS biosynthesis had long been postulated to be a prerequisite for iduronic acid (IdoUA) formation by C5-epimerization of GlcUA. This hypothesis has recently been argued based on enzymological studies using microsomes that C5-epimerization precedes 4-O-sulfation, which was further supported by the specificity of the cloned D4ST-1 with predominant preference for IdoUA-GalNAc flanked by GlcUA-GalNAc over IdoUA-GalNAc flanked by IdoUA-GalNAc in exhaustively desulfated dermatan. Whereas the counterproposal explains the initial reactions, apparently it cannot rationalize the synthetic mechanism of IdoUA-GalNAc(4-O-sulfate)-rich clusters typical of mature DS chains. In this study, we examined detailed specificities of the three recombinant human 4-O-sulfotransferases using partially desulfated DS as an acceptor. Enzymatic analysis of the transferase reaction products showed that D4ST-1 far more efficiently transferred sulfate to GalNAc residues in -IdoUA-Gal-NAc-IdoUA-than in -GlcUA-GalNAc-GlcUA-sequences. In contrast, C4ST-1 showed the opposite preference, and C4ST-2 used GalNAc residues in both sequences to comparable degrees, being consistent with its phylogenetic relations to D4ST-1 and C4ST-1. Structural analysis of the oligosaccharides, which were isolated after chondroitinase AC-I digestion of the 35S-labeled transferase reaction products, revealed for the first time that D4ST-1, as compared with C4ST-1 and C4ST-2, most efficiently utilized GalNAc residues located not only in the sequence -IdoUA-GalNAc-IdoUA- but also in -GlcUA-Gal-NAc-IdoUA- and -IdoUA-GalNAc-GlcUA-. The isolated oligosaccharide structures also suggest that 4-O-sulfation promotes subsequent 4-O-sulfation of GalNAc in the neighboring disaccharide unit.     

Expression of sulfotransferases involved in the biosynthesis of chondroitin sulfate E in the bone marrow derived mast cells

Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with 35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.     

Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. We previously reported the cloning of C4ST cDNA from mouse brain. We here report the cloning and expression of human C4ST cDNA. The cDNA was isolated from a human fetal brain cDNA library by hybridization with a DNA probe prepared from rat poly(A)(+) RNA used for the cloning of mouse C4ST cDNA. The cDNA comprises a single open reading frame that predicts a Type II transmembrane protein composed of 352 amino acids. The protein has an amino acid sequence homology of 96% with mouse C4ST. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that transfers sulfate to both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis indicated that human C4ST mRNAs (6.0 and 1.9 kb) are expressed ubiquitously in various adult human tissues. Dot blot analysis has shown that human C4ST is strongly expressed in colorectal adenocarcinoma and peripheral blood leukocytes, whereas strong expression of human chondroitin 6-sulfotransferase (C6ST) is observed in aorta and testis. These observations suggest that the expression of C4ST and C6ST may be controlled differently in human tissues. The C4ST gene was localized to chromosome 12q23.2-q23.3 by fluorescence in situ hybridization.     

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.     

Synthesis of sulfated phenyl 2-acetamido-2-deoxy-D-galactopyranosides. 4-O-Sulfated phenyl 2-acetamido-2-deoxy-beta-D-galactopyranoside is a competitive acceptor that decreases sulfation of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase

We have previously cloned N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 hydroxyl group of the GalNAc 4-sulfate residue of chondroitin sulfate A and forms chondroitin sulfate E containing GlcA-GalNAc(4,6-SO(4)) repeating units. To investigate the function of chondroitin sulfate E, the development of specific inhibitors of GalNAc4S-6ST is important. Because GalNAc4S-6ST requires a sulfate group attached to the C-4 hydroxyl group of the GalNAc residue as the acceptor, the sulfated GalNAc residue is expected to interact with GalNAc4S-6ST and affect its activity. In this study, we synthesized phenyl alpha- or -beta-2-acetamido-2-deoxy-beta-D-galactopyranosides containing a sulfate group at the C-3, C-4, or C-6 hydroxyl groups and examined their inhibitory activity against recombinant GalNAc4S-6ST. We found that phenyl beta-GalNAc(4SO(4)) inhibits GalNAc4S-6ST competitively and also serves as an acceptor. The sulfated product derived from phenyl beta-GalNAc(4SO(4)) was identical to phenyl beta-GalNAc(4,6-SO(4)). These observations indicate that derivatives of beta-D-GalNAc(4SO(4)) are possible specific inhibitors of GalNAc4S-6ST.     

The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.     

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

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO(4)) 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(4SO(4)) residues of chondroitin sulfate A (CS-A); nevertheless, the nonreducing terminal GalNAc(4SO(4)) could be sulfated efficiently when the GalNAc(4SO(4)) residue was included in the unique nonreducing terminal structure, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), 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(4SO(4)) residues included in a tetrasaccharide sequence, GlcA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), were sulfated efficiently by squid GalNAc4S-6ST, and the E-D hybrid tetrasaccharide sequence, GlcA-GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) 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.     

Chondroitin 4-O-sulfotransferase-2 regulates the number of chondroitin sulfate chains initiated by chondroitin N-acetylgalactosaminyltransferase-1

Recently, it has been shown that a deficiency in ChGn-1 (chondroitin N-acetylgalactosaminyltransferase-1) reduced the numbers of CS (chondroitin sulfate) chains, leading to skeletal dysplasias in mice. Although these results indicate that ChGn-1 regulates the number of CS chains, the mechanism mediating this regulation is not clear. ChGn-1 is thought to initiate CS biosynthesis by transferring the first GalNAc (N-acetylgalactosamine) to the tetrasaccharide in the protein linkage region of CS. However, in vitro chondroitin polymerization does not occur on the non-reducing terminal GalNAc-linkage pentasaccharide structure. In the present study we show that several different heteromeric enzyme complexes composed of different combinations of four chondroitin synthase family members synthesized more CS chains when a GalNAc-linkage pentasaccharide structure with a non-reducing terminal 4-O-sulfation was the CS acceptor. In addition, C4ST-2 (chondroitin 4-O-sulfotransferase-2) efficiently transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of non-reducing terminal GalNAc-linkage residues, and the number of CS chains was regulated by the expression levels of C4ST-2 and of ChGn-1. Taken together, the results of the present study indicate that C4ST-2 plays a key role in regulating levels of CS synthesized via ChGn-1.     

Expression of chondroitin-4- O -sulfotransferase in Escherichia coli and Pichia pastoris

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.