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.     

Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.     

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.     

A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO4)). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO4) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) because GalNAc(4SO4) and deltaHexA(2SO4)-GalNAc(6SO4) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO4) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO4)-GlcAGalNAc(6SO4). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [35S]PAPS and GalNAc4S-6ST and the 35S-labeled product was digested with chondroitinase ACII, a 35S-labeled trisaccharide (Oligo III) containing [35S]GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.     

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO₄)) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO₄) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO₄) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO₄) residues in both CS and DS. IdoA-GalNAc(4,6-SO₄) units that account for ∼40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO₄) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO₄) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.     

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.     

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.     

Chemical synthesis of 4-azido-β-galactosamine derivatives for inhibitors of <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine 4-sulfate 6-<Emphasis Type="Italic">O</Emphasis>-sulfotransferase

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-β-GalNAc derivatives and 4-azido-β-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-β-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC₅₀ value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.     

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.     

Inhibition of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase by ß-D-4-O-sulfo-N-acetylgalactosaminides bearing various hydrophobic aglycons

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO₄) residues of chondroitin sulfate to yield chondroitin sulfate E (CS-E). We have previously demonstrated that phenyl-ß-D-GalNAc(4SO₄) could serve as an acceptor for GalNAc4S-6ST, thereby inhibiting GalNAc4S-6ST competitively. In this paper we compared the inhibitory effects of various glycosides in which various hydrophobic aglycons were attached to D-GalNAc(4SO₄) via ß anomeric configuration. p-Nitrophenyl-ß-D-GalNAc(4SO₄) and p-chlorophenyl-ß-D-GalNAc(4SO₄) were stronger inhibitors than phenyl-ß-D-GalNAc(4SO₄). Among inhibitors examined here, 3-estradiol-ß-D-GalNAc(4SO₄) was the strongest inhibitor; the Ki of 3-estradiol-ß-D-GalNAc(4SO₄) for the competitive inhibition was 0.008 mM, which was much lower than the Ki of phenyl-ß-D-GalNAc(4SO₄), 0.98 mM. In contrast, 7-estradiol-ß-D-GalNAc(4SO₄) showed only weak inhibition to GalNAc4S-6ST. 3-Estradiol- ß-D-GalNAc(4SO₄) did not inhibit chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase under the concentration where GalNAc4S-6ST was inhibited by 90%. When 3-estradiol- ß-D-GalNAc(4SO₄) was added to the culture medium of chondrosarcoma cells expressing human GalNAc4S-6ST, a significant, albeit small, reduction in the cellular synthesis of CS-E was observed. These results suggest that estradiol group of 3-estradiol-ß-D-GalNAc(4SO₄) may enhance the inhibitory activity of the glycoside through increasing the affinity to the enzyme and may allow the glycosides to diffuse at a low efficiency into the cells to inhibit cellular synthesis of CS-E.     

Recognition of sulfation pattern of chondroitin sulfate by uronosyl 2-O-sulfotransferase

We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [(35)S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [(35)S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be DeltaHexA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO(4))-GlcA-GalNAc(6SO(4))-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) and GlcA-GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.     

A terminal 6-sulfotransferase catalyzing a synthesis of N-acetylgalactosamine 4,6-bissulfate residue at the nonreducing terminal position of chondroitin sulfate

A soluble enzyme from quail oviduct which incorporates sulfate into position 6 of the nonreducing N-acetylgalactosamine 4-sulfate end group of chondroitin sulfate has been purified. This enzyme (termed "terminal 6-sulfotransferase") was partially separated from a 6-sulfotransferase present in the same tissue which catalyzes the incorporation of sulfate into interior portion of unsulfated chondroitin. The basic requirements for the terminal 6-sulfotransferase reaction were shown to be 3'-phosphoadenylyl sulfate (donor) and chondroitin 4-sulfate (acceptor). The substitution of unsulfated chondroitin (prepared from squid skin) for chondroitin 4-sulfate resulted in a total loss of activity. These results suggest that the organization of the proteoglycan-synthesizing apparatus may well involve hitherto unrecognized mechanisms for the sulfation of chondroitin chains.     

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.     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.     

Difference between N-acetylgalactosamine 4-sulfate 6-O-sulfotransferases from human serum and squid cartilage in specificity toward the terminal and interior portion of chondroitin sulfate

In the preceding paper (Inoue, H., Otsu, K., Yoneda, M., Kimata, K., Suzuki, S., and Nakanishi, Y. (1986) J. Biol. Chem. 261, 4460-4469), we reported the purification from human serum of an N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase fraction which was able to transfer sulfate predominantly to position 6 of the nonreducing terminal N-acetylgalactosamine 4-sulfate unit of chondroitin sulfate. We now show that the activity toward the terminal was co-purified with a minor activity toward the interior counterpart by sequential chromatography on heparin-Sepharose CL-6B, Matrex Blue B, hydroxyapatite, and Sephacryl S-300, and that the two activities were equally heatlabile. The enzyme purified 5000-fold from human serum was devoid of the sulfotransferase activities toward chondroitin, heparan sulfate, and keratan sulfate, but showed a strong terminal sulfotransferase activity toward dermatan sulfate (pig skin); over 97% of the sulfate residues incorporated were at position 6 of the nonreducing N-acetylgalactosamine 4,6-bissulfate end groups linked to the L-iduronic acid group. Although the enzyme introduces sulfate predominantly into the nonreducing terminal of chondroitin sulfate at physiological pH (approximately equal to 7.0) and Ca2+ concentration (approximately 2-3 mM), the activity toward the interior portion relative to that toward the terminal was increased by either lowering pH or elevating Ca2+ concentration, perhaps owing to changes in the conformation or ionic state of the acceptor molecule. Comparison between the human serum enzyme and the N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (formerly designated "E6-sulfotransferase") from squid cartilage indicated that the latter is distinct from the former in introducing sulfate predominantly into the interior portion of chondroitin sulfate. It appears that the role of the squid sulfotransferase is to synthesize so-called chondroitin sulfate E where over 50% of the interior hexosamine units are 4,6-bis-sulfated.