New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

A model of the platelet factor 4 complex with heparin.

A model of heparin bound to bovine platelet factor 4 (BPF4) was completed using a graphically designed heparin molecule and the crystallographic coordinates of the native bovine platelet factor 4 tetramer. The oligosaccharides had a chain length of at least eight disaccharide units with the major repeating disaccharide unit consisting of (1----4)-O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1----4)-(2-deoxy-2-sulfamino-2-D-glucopyranosyl 6-sulfate). Each disaccharide unit carried a -4.0 charge. The structure of BPF4 was solved to 2.6 A resolution with R = 0.237. Each monomer of BPF4 contains an alpha-helix lying across 3 strands of antiparallel beta-sheet. Each helix has four lysines, which have been implicated in heparin binding. These lysine residues are predominantly on one side of the helix and are solvent accessible. Electrostatic calculations performed on the BPF4 tetramer show a ring of strong, positive charge which runs perpendicularly across the helices. Included in this ring of density is His-38, which has been shown by NMR to have a large pKa shift when heparin binds to BPF4. Our model of heparin bound to PF4 has the anionic polysaccharide perpendicular to the alpha-helices, wrapped about the tetramer along the ring of positive charge, and salt linked to all four lysines on the helix of each monomer.     

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO₂. The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05–2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.     

Use of biosynthetic enzymes in heparin and heparan sulfate synthesis

Heparan sulfate and heparin are highly sulfated polysaccharides consisting of repeating disaccharide units of glucuronic acid or iduronic acid that is linked to glucosamine. Heparan sulfate displays a range of biological functions, and heparin is a widely used anticoagulant drug in hospitals. It has been known to organic chemists that the chemical synthesis of heparan sulfate and heparin oligosaccharides is extremely difficult. Recent advances in the study of the biosynthesis of heparan sulfate/heparin offer a chemoenzymatic approach to synthesize heparan sulfate and heparin. Compared to chemical synthesis, the chemoenzymatic method shortens the synthesis and improves the product yields significantly, providing an excellent opportunity to advance the understanding of the structure and function relationships of heparan sulfate. In this review, we attempt to summarize the progress of the chemoenzymatic synthetic method and its application in heparan sulfate and heparin research.     

Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.     

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (GlcpA) and idopyranosyluronic acid (IdopA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (GlcpNAc) residue was lost on workup. The GlcpA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6-O-sulfo glycopyranosylamine (GlcpNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in GlcpNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained GlcpNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid <Emphasis Type="Italic">Ommastrephes bartrami</Emphasis> by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: -[3GlcAbeta1-4(GalNAcalpha1-3)Fucalpha1](n)-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique (0,2)A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position-3 of the disaccharide repeat.     

Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).     

Degradation of even-numbered reduced and non-reduced hyaluronate oligosaccharides with D-glucuronic acid or N-acetyl-D-glucosamine as non-reducing terminal by chondroitin ABC and AC lyases.

Chondroitin ABC and AC lyases split hexosaminidic linkages in galactosaminoglycans and hyaluronic acid. Even-numbered oligosaccharides from hyaluronic acid with either D-glucuronic acid or N-acetylglucosamine in non-reducing position were used, prior to and after reduction with sodium borohydride, as substrates for chondroitin ABC and AC lyases. These substrates allowed elucidation of the effects of the nearest neighborhood of the bond to be split on the action of the enzymes. The results indicate that chondroitin ABC lyase acts strictly as an endolyase towards hyaluronate and requires the presence of a disaccharide in both reducing and non-reducing positions of the endohexosaminidic bond to be split. None of the hexosaminidic bonds of the tetrasaccharide GlcNAc-GlcUA-GlcNAc-GlcUA is split by chondroitin ABC lyase. In contrast chondroitin AC lyase acts also as an exoglycosidase towards hyaluronate and recognizes only the amino sugar and the uronic acid residue that are linked via the hexosaminidic bond which is split. Thus, the N-acetylglucosamine and glucuronic acid residues at both ends of a tetrasaccharide with the structure GlcNAc-GlcUA-GlcNAc-GlcUA are liberated.     

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.     

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.     

Identification of N-sulphated disaccharide units in heparin-like polysaccharides.

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.     

The determination of iduronic acid and glucuronic acid in heparins by a new technique: Re-evaluation of the variable hexuronic acid composition of mammalian heparins

A new technique for the quantitative determination of the uronic acid components of heparin is described. Heparin was deamination with organic nitrite and methanolyzed. The monomeric derivatives of uronic acids were quantitated by gas chromotography. Depolymerization to monomeric units appeared essentially complete as judged from the yield of uronic acid derivatives. By applying the method the relative contents of iduronic acid and glucuronic acid in a number of heparin samples were estimated. In all samples examined the content of iduronic acid was larger than that of glucuronic acid. A species specific difference in the iduronic acid to glucuronic acid ration of heparin was noted. Noticeable difference of this ratio was observed also between different organs of a species of animal and among heparin fractions obtained from an organ.     

Structures of heparin-derived disaccharide bound to cobra cardiotoxins: context-dependent conformational change of heparin upon binding to the rigid core of the three-fingered toxin

Glycosaminoglycans (GAGs) have been suggested to be a potential target for cobra cardiotoxin (CTX) with high affinity and specificity via a cationic belt at the concave surface of the polypeptide. The interaction of GAGs, such as high-molecular weight heparin, with CTXs not only can induce aggregation of CTX molecules but also can enhance their penetration into membranes. The binding of short chain heparin, such as a heparin-derived disaccharide [DeltaUA2S(1-->4)-alpha-D-GlcNS6S], to CTX A3 from Taiwan cobra (Naja atra), however, will not induce aggregation and was, therefore, investigated by high-resolution (1)H NMR. A novel heparin binding site on the convex side of the CTX, near the rigid disulfide bond-tightened core region of Cys38, was identified due to the observation of intermolecular NOEs between the protein and carbohydrate. The derived carbohydrate conformation using complete relaxation and conformational exchange matrix analysis (CORCEMA) of NOEs indicated that the glycosidic linkage conformation and the ring conformation of the unsaturated uronic acid in the bound state depended significantly on the charge context of CTX molecules near the binding site. Specifically, comparative binding studies of several heparin disaccharide homologues with two CTX homologues (CTX Tgamma from Naja nigricollis and CTX A3) indicated that the electrostatic interaction of N-sulfate of glucosamine with NH(3)(+)zeta of Lys12 and of the 2-O-sulfate of the unsaturated uronic acid with NH(3)(+)zeta of Lys5 played an important role. These results also suggest a model on how the CTX-heparin interaction may regulate heparin-induced aggregation of the toxin via the second heparin binding site.     

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.     

Chondroitin C lyase [4.2.2.] is unable to cleave fructosylated sequences inside the partially fructosylated <Emphasis Type="Italic">Escherichia coli</Emphasis> K4 polymer

Chondroitin C lyase was demonstrated to be unable to act on fructosylated sequences inside a partially fructosylated polysaccharide having the chondroitin backbone structure, the Escherichia coli K4 polymer, using different analytical approaches. Chondroitin C lyase produced various unsaturated oligosaccharides by acting on an approximately 27%-fructosylated K4 polymer. The online HPLC-ESI-MS approach showed the disaccharide nature of the main species produced by chondroitinase C as DeltaHexA-GalNAc. Furthermore, the non-digested sequences inside the K4 polymer were demonstrated to be oligosaccharides bearing a fructose for each glucuronic acid unit. In fact, unsaturated fully fructosylated oligomers, from tetrasaccharide to decasaccharide (DeltaHexA(Fru)-GalNAc-[GlcA(Fru)-GalNAc](n) with n between 1 and 4), at decreasing percentages, were produced by the enzyme. These results clearly indicate that chondroitinase C cleaved the innermost glucuronic acid-N-acetylgalactosamine linkage without affecting the 1,4 glycosidic linkage between fructosylated glucuronic acid and N-acetylgalactosamine residues, confirming that the 3-O-fructosylation of the GlcA residue renders the polysaccharide resistant to the enzyme action. This novel specific activity of chondroitinase C was also useful for the production of discrete microgram amounts of fully fructosylated oligomers, from 4- to 10-mers, from E. coli K4 for possible further studies and applications.     

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide

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

The disaccharide repeating-units of heparan sulfate

Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.     

Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product

Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.30 A resolution. The protein is composed of three domains: an N-terminal alpha-helical domain, a central two-layered beta-sheet domain, and a C-terminal domain forming a two-layered beta-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "+1" and "+2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.