Role of the carboxyl-terminal region in the activity of N-acetylglucosamine 6-o-sulfotransferase-1

N-Acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 position of non-reducing N-acetylglucosamine. N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) is the first cloned GlcNAc6ST and is involved in the synthesis of the L-selectin ligand. We noticed conserved C-terminal segments among GlcNAc6STs and produced mutant enzymes to reveal the functional significance. Mutant enzymes were transiently expressed as fusion proteins with protein A in COS-7 cells, and some of them were purified to homogeneity by IgG Sepharose column chromatography. Deletion of a C-terminal segment (amino acid numbers 479-483) resulted in a complete loss of the activity, when assayed using GlcNAcbeta1-6ManOMe as a substrate. Upon site-directed mutagenesis of the C-terminal region, three mutants, L477A, L478A and L483A, exhibited reduced activity. The K(M )values for GlcNAcbeta1-6ManOMe of L477A and L478A were 4 times higher than the K(M) of the wild-type enzyme, while that of L483A was unchanged. On the other hand the K(M )for PAPS of L483A was 3 times higher than that of the wild-type enzyme, while the values of L477A and L478A were unchanged. Furthermore, the L477A mutant acted on a core 3 structure (GlcNAcbeta1-3GalNAc-pNP), while the wild-type enzyme does not. These results demonstrate a role for leucine residues in the C-terminal region in the enzymatic activity.     

Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3'-phosphate,5'-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcbeta1-6[Galbeta1-3]GalNAc-pNP (core 2), GlcNAcbeta1-6ManOMe, and GlcNAcbeta1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcbeta1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919-929).     

N-acetylglucosamine-6-O-sulfotransferase-1: production in the baculovirus system and its applications to the synthesis of a sulfated oligosaccharide and to the modification of oligosaccharides in fibrinogen

N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the C-6 position of non-reducing GlcNAc. Human GlcNAc6ST-1 was expressed as a fusion protein with protein A in an insect cell line (Tn 5 cells) using the baculovirus system. The recombinant enzyme was purified to homogeneity by IgG Sepharose column chromatography. The substrate specificity and the kinetic properties of the enzyme were similar to those of the enzyme expressed in the mammalian system. The purified recombinant enzyme was used to synthesize 6-sulfo GlcNAcbeta1-3Galbeta1-4Glc, which was identified by time of flight mass spectrometry. This sulfated trisaccharide served as a better substrate for microsomal galactosyltransferase from the mouse colon compared to 6-sulfo GlcNAc. The purified recombinant enzyme was also used to sulfate oligosaccharide chains on fibrinogen after enzymatic desialylation and degalactosylation to expose nonreducing GlcNAc residues. It is known that desialylation greatly increases the rate of clotting of fibrinogen after the addition of thrombin. Subsequent sulfation of desialylated and degalactosylated fibrinogen slightly decreased the rate of clotting. The recombinant GlcNAc6ST-1 is a useful reagent for 6-sulfate exposed GlcNAc residues both in oligosaccharides and in glycoproteins.     

Combinatorial PCR approach to homology-based cloning: Cloning and expression of mouse and human GM3-synthase

GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.     

Enzymatic synthesis in vitro of the disulfated disaccharide unit of corneal keratan sulfate

Among the enzymes of the carbohydrate sulfotransferase family, human corneal GlcNAc 6-O-sulfotransferase (hCGn6ST, also known as human GlcNAc6ST-5/GST4beta) and human intestinal GlcNAc 6-O-sulfotransferase (hIGn6ST or human GlcNAc6ST-3/GST4alpha) are highly homologous. In the mouse, intestinal GlcNAc 6-O-sulfotransferase (mIGn6ST or mouse GlcNAc6ST-3/GST4) is the only orthologue of hCGn6ST and hIGn6ST. In the previous study, we found that hCGn6ST and mIGn6ST, but not hIGn6ST, have sulfotransferase activity to produce keratan sulfate (Akama, T. O., Nakayama, J., Nishida, K., Hiraoka, N., Suzuki, M., McAuliffe, J., Hindsgaul, O., Fukuda, M., and Fukuda, M. N. (2001) J. Biol. Chem. 276, 16271-16278). In this study, we analyzed the substrate specificities of these sulfotransferases in vitro using synthetic carbohydrate substrates. We found that all three sulfotransferases can transfer sulfate to the nonreducing terminal GlcNAc of short carbohydrate substrates. Both hCGn6ST and mIGn6ST, but not hIGn6ST, transfer sulfate to longer carbohydrate substrates that have poly-N-acetyllactosamine structures, suggesting the involvement of hCGn6ST and mIGn6ST in production of keratan sulfate. To clarify further the involvement of hCGn6ST in biosynthesis of keratan sulfate, we reconstituted the biosynthetic pathway in vitro by sequential enzymatic treatment of a synthetic carbohydrate substrate. Using four enzymes, beta1,4-galactosyltransferase-I, beta1,3-N-acetylglucosaminyltransferase-2, hCGn6ST, and keratan sulfate Gal 6-O-sulfotransferase, we were able to synthesize in vitro a product that conformed to the basic structural unit of keratan sulfate. Based on these results, we propose a biosynthetic pathway for N-linked keratan sulfate on corneal proteoglycans.     

Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate

Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.     

KSGal6ST Is Essential for the 6-Sulfation of Galactose within Keratan Sulfate in Early Postnatal Brain

Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.     

Genetically altered mice with different sialyltransferase deficiencies show tissue-specific alterations in sialylation and sialic acid 9-O-acetylation

Glycan chains on glycoconjugates traversing the Golgi apparatus are often terminated by sialic acid residues, which can also be 9-O-acetylated. This process involves competition between multiple Golgi enzymes. Expression levels of Golgi enzyme mRNAs do not always correlate with enzyme activity, which in turn cannot accurately predict glycan sequences found on cell surfaces. Here we examine the cell type-specific expression of terminal glycans in tissues of normal mice in comparison with animals deficient in ST6Gal-I (transfers alpha2-6-linked sialic acid to Galbeta1-4GlcNAc) or ST3Gal-I (transfers alpha2-3-linked sialic acid to Galbeta1-3GalNAc). Tissues of ST6Gal-I null mice showed minimal binding of an alpha2-6-sialic acid-specific lectin, indicating that no other enzyme generates Siaalpha2-6Galbeta1-4GlcNAc and that Siaalpha2-6GalNAc (sialyl-Tn) is rare in mice. However, exposed Galbeta1-4GlcNAc termini were only moderately increased, indicating that these can be partially capped by other enzymes. Indeed, Galalpha1-3Galbeta1-4GlcNAc and Fucalpha1-2Galbeta1-4GlcNAc termini were enhanced in some tissues. Many tissues of ST3Gal-I null animals showed increases in Galbeta1-3GalNAc termini, and some increases in poly-N-acetyllactosamines. However, overall expression of alpha2-3-linked sialic acid was selectively reduced only in a few instances, indicating that other ST3Gal enzymes can generate this linkage in most tissues. Highly selective losses of 9-O-acetylation of sialic acid residues were also observed, with ST6Gal-I deficiency causing loss on endothelium and ST3Gal-I deficiency giving a marked decrease on CD4(+) lymphocytes. These data demonstrate selective regulation of sialylation and 9-O-acetylation, point to cell types with potential physiological defects in null animals, and show in vivo evidence for competition between Golgi enzymes.     

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase expression during early mouse embryonic development

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is an enzyme which is known to help build up the GlcAbeta1-3GalNAc(4,6-bisSO4) unit of chondroitin sulfate E (CS-E). This enzymatic activity has been reported in squid cartilage and in human serum, but has never been reported as an enzyme required during early mouse development. On the other hand, CS-E has been shown to bind with strong affinity to Midkine (MK). The latter is a heparin-binding growth factor which has been found to play important regulatory roles in differentiation and morphogenesis during mouse embryonic development. We have analyzed the expression pattern of the GalNAc4S-6ST gene during early mouse embryonic development by whole mount in situ hybridization. The results show that GalNAc4S-6ST is differentially expressed in the anterior visceral ectoderm at stage E5.5 and later becomes restricted to the embryonic endoderm, especially in the prospective midgut region. During the turning process, expression of GalNAc4S-6ST gene is detected in the forebrain, branchial arches, across the gut tube (hindgut, midgut and foregut diverticulum), in the vitelline veins and artery and in the splanchnopleure layer. These results open the possibility of a role for GalNAc4S-6ST during early mouse development.     

Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl-oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I-IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT-PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STS: Furthermore, to assay activity-related changes in ST expression, we used amygdaloid-kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up-regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down-regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.     

Functional expression and genomic structure of human N-acetylglucosamine-6-O-sulfotransferase that transfers sulfate to beta-N-acetylglucosamine at the nonreducing end of an N-acetyllactosamine sequence

The cDNA and gene encoding human N-acetylglucosamine-6-O-sulfotransferase (Gn6ST) have been cloned. Comparative analysis of this cDNA with the mouse Gn6ST sequence indicates 96% amino acid identity between the two sequences. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase, which transferred sulfate to the terminal GlcNAc in GlcNAcbeta1-O-CH(3), GlcNAcbeta1-3Galbeta1-O-CH(3) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc but not in GlcNAcalpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4 Xylbeta1-O-Ser. In addition, neither Galbeta1-4GlcNAcbeta1-O-naphthalenemethanol nor GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4X ylbeta1-O-Ser were utilized as acceptors. These findings indicate that a terminal beta-linked GlcNAc residue is necessary for acceptor substrates of Gn6ST. The human Gn6ST gene spans about 7 kb, consists of two exons and exhibits an intron-less coding region.     

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.     

Comparison of the enzymatic properties of mouse beta-galactoside alpha2,6-sialyltransferases,ST6Gal I and II

The cDNA encoding a second type of mouse beta-galactoside alpha2,6-sialyltransferase (ST6Gal II) was cloned and characterized. The sequence of mouse ST6Gal II encoded a protein of 524 amino acids and showed 77.1% amino acid sequence identity with human ST6Gal II. Recombinant ST6Gal II exhibited alpha2,6-sialyltransferase activity toward oligosaccharides that have the Galbeta1,4GlcNAc sequence at the nonreducing end of their carbohydrate groups, but it exhibited relatively low and no activity toward some glycoproteins and glycolipids, respectively. On the other hand, ST6Gal I, which has been known as the sole member of the ST6Gal-family for more than ten years, exhibited broad substrate specificity toward oligosaccharides, glycoproteins, and a glycolipid, paragloboside. The ST6Gal II gene was mainly expressed in brain and embryo, whereas the ST6Gal I gene was ubiquitously expressed, and its expression levels were higher than those of the ST6Gal II gene. The ST6Gal II gene is located on chromosome 17 and spans over 70 kb of mouse genomic DNA consisting of at least 6 exons. The ST6Gal II gene has a similar genomic structure to the ST6Gal I gene. In this paper, we have shown that ST6Gal II is a counterpart of ST6Gal I.     

Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II.

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.     

Expression of long-form N-acetylglucosamine-6-O-sulfotransferase 1 in human high endothelial venules

Two members of the N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) family, GlcNAc6ST-1 and GlcNAc6ST-2, function in the biosynthesis of 6-sulfo sialyl Lewis X-capped glycoproteins expressed on high endothelial venules (HEVs) in secondary lymphoid organs. Thus, both enzymes play a critical role in L-selectin-expressing lymphocyte homing. Human GlcNAc6ST-1 is encoded by a 1593-bp open reading frame exhibiting two 5' in-frame methionine codons spaced 141 bp apart. Both resemble the consensus sequence for translation initiation. Thus, it has been hypothesized that both long and short forms of GlcNAc6ST-1 may be present, although endogenous expression of either form has not been confirmed in humans. Here, the authors developed an antibody recognizing amino acid residues between the first two human GlcNAc6ST-1 methionines. This antibody specifically recognizes the long form of the enzyme, a finding validated by Western blot analysis and immunofluorescence cytochemistry of HeLa cells misexpressing long and/or short forms of human GlcNAc6ST-1. Using this antibody, the authors carried out immunofluorescence histochemistry of human lymph node tissue sections and found endogenous expression of the long form of the enzyme in human tissue, predominantly in the trans-Golgi network of endothelial cells that form HEVs.     

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.