The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans.

Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and keratan sulphate were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0, and the resulting products were reduced with NaB3H4. This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues, which were converted into 3H-labelled 2,5-anhydro-D-mannitol (AManR) or 2,5-anhydro-D-talitol (ATalR) residues respectively. The end-labelled disaccharides, composed of D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-galactose (Gal) and one of the anhydrohexitols, were identified as follows: both chondroitin 4-sulphate and chondroitin 6-sulphate gave GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4), IdoA----ATalR (4-SO4) and GlcA(2-SO4)----ATalR(6-SO4); dermatan sulphate gave IdoA----ATalR(4-SO4), GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4)----IdoA(2-SO4)ATalR(4-SO4) and IdoA----ATalR (4,6-diSO4); keratan sulphate gave Gal(6-SO4)----AManR(6-SO4), Gal----AManR(6-SO4), Gal(6-SO4)----AManR and Gal----AManR. Several additional disaccharides were generated by treatment of the uronic acid-containing disaccharides with hydrazine to epimerize their uronic acid residues at C-5. A number of these disaccharides were found to be substrates for lysosomal sulphatases and glycuronidases. Methods were developed for the separation of all of the disaccharide products by h.p.l.c. The rate of N-deacetylation of chondroitin 4-sulphate by hydrazinolysis was significantly lower than the rate of N-deacetylation of chondroitin 6-sulphate or chondroitin. Dermatan sulphate was N-deacetylated at an intermediate rate. The relative amounts of disaccharides obtained from chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate under optimum hydrazinolysis/deamination conditions were comparable with the amounts of the corresponding products released from the polymers by chondroitinase treatment.     

Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing approximately 2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)----ATalR(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.     

Changes in the structure and biological property of N----O sulfate-transferred, N-resulfated heparin

The N----O sulfate transfer of heparin has been investigated as an approach to chemical 3-O-sulfation of the D-glucosamine residues in heparin. The pyridinium salt of porcine heparin was heated at 90 degrees C in solid state for 90 min (in vacuo over P2O5) to effect the transfer of the N-sulfate groups to the HO groups in the polysaccharide, followed by N-resulfation. The product (N----O sulfate-transferred, N-resulfated heparin (ST heparin] was depolymerized with HONO to generate a mixture of di- and higher oligosaccharides. The borohydride-reduced oligosaccharides were separated on Bio-Gel P-4 and DEAE-Sephacel. The disaccharide trisulfate fraction (10.4% yield) was found to be a mixture of nearly equal amounts of IdoA(2-SO4)-AManR(3,6-diSO4) and IdoA(2,3-diSO4)-AManR(6-SO4), where IdoA represents L-iduronic acid and AManR represents the alditol formed by reduction of 2,5-anhydro-D-mannose with NaBH4. Chemical and NMR spectroscopic analyses revealed that the N----O sulfate transfer proceeded preferentially at HO-3 positions in both 6-O-sulfo-D-glucosamine and 2-O-sulfo-L-iduronic acid residues. Chromatography on antithrombin III-Sepharose gel indicated that the structural change involved in ST heparin resulted in an obvious increase in the ability to bind antithrombin III. Biological examination also indicated that this structural change resulted in moderate increases in all the activities (blood anti-clotting, anti-Factor IIa, and anti-Factor Xa) and in the strength of intrinsic fluorescence of antithrombin III.     

Transport of heparan sulfate into the nuclei of hepatocytes

Monolayer cultures of a rat hepatocyte cell line shown previously to accumulate a nuclear pool of free heparan sulfate chains that are enriched in sulfated glucuronic acid (GlcA) residues (Fedarko, N.S., and Conrad, H.E., (1986) J. Cell Biol. 587-599) were incubated with 35SO4(2-), and the rate of appearance of heparan [35S]sulfate in the nuclei was measured. Heparan [35S]sulfate began to accumulate in the nuclei 2 h after the administration of 35SO4(2-) to the cells and reached a steady state level after 20 h. Heparan [35S]sulfate was lost from the nuclei of prelabeled cells with a t1/2 of 8 h. Chloroquine did not inhibit the transport of heparan sulfate into the nucleus, but increased the t1/2 for the exit of heparan sulfate from the nucleus to 20 h and led to a doubling of the steady state level of nuclear heparan sulfate. Heparan [35S]sulfate which was obtained from the medium or from the cell matrix of a labeled culture and which contained only low levels of GlcA-2-SO4 residues was incubated with cultures of unlabeled cells, and the uptake of the exogenous heparan [35S]sulfate was studied. At 37 degrees C the cells took up proteoheparan [35S]sulfate and transported about 10% of the internalized heparan [35S]sulfate into the nucleus, where it appeared as free chains. The heparan [35S]sulfate isolated from the nucleus was enriched in GlcA-2-SO4 residues, whereas the heparan [35S]sulfate remaining in the rest of the intracellular pool showed a corresponding depletion in GlcA-2-SO4 residues. At 16 degrees C, where endocytosed materials do not enter the lysosomes, the cells also transported exogenous proteoheparan [35S]sulfate to the nucleus with similar processing. Thus, the metabolism of exogenous heparan sulfate by hepatocytes follows the same pathway observed in continuously labeled cells and does not involve lysosomal processing of the internalized heparan sulfate.     

Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins.

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.     

Characterization of novel sequences containing 3-O-sulfated glucosamine in glomerular basement membrane heparan sulfate and localization of sulfated disaccharides to a peripheral domain

Fragmentation of the heparan sulfate chains from bovine glomerular basement membrane (GBM) by hydrazine/nitrous acid treatment followed by NaB3H4-reduction yielded a mixture of six sulfated disaccharides containing D-glucuronic (GlcUA) or L-iduronic acid (IdUA) and terminating in 2,5-anhydro[3H]mannitol (AnManH2), in addition to the nonsulfated component GlcUA beta 1----4AnManH2. Among these products two novel disaccharide units were identified as IdUA alpha 1----4AnManH2(3-SO4) and IdUA(2-SO4)alpha 1----4AnManH2(3-SO4); these accounted for 22% of the total sulfated species indicating that there are 2-3 residues of 3-O-sulfated glucosamine/heparan sulfate chain. The disulfated disaccharide was shown through its release by direct nitrous acid treatment to be situated in a GlcNSO3-IdUA(2-SO4)-GlcNSO3(3-SO4) sequence which is distinct from that in which 3-O-sulfated glucosamine is located in the antithrombin-binding region of heparins. Analyses of heparan sulfate from lens capsule, a nonvascular basement membrane, indicated the absence of sequences containing 3-O-sulfated glucosamine, although otherwise the sulfated disaccharides produced by hydrazine/nitrous acid/Na-B3H4 treatment (GlcUA beta 1----4AnManH2(6-SO4), IdUA alpha 1----4AnManH2(6-SO4), IdUA(2-SO4)alpha 1----4AnManH2 and IdUA(2-SO4)alpha 1----4AnManH2(6-SO4] were the same as from GBM. Examination of the GBM heparan sulfate domains after nitrous acid treatment indicated that the O- as well as N-sulfate groups are clustered in an iduronic acid-rich 10-disaccharide peripheral segment, while the internal region (approximately 20 disaccharides) is composed primarily of repeating GlcUA beta 1----4GlcNAc units. The localization of chain diversity to the outer region may facilitate interactions of the heparan sulfate with other macromolecular components.     

A novel sulfated structure in the carbohydrate-protein linkage region isolated from porcine intestinal heparin.

A preparation of porcine stage 14 intestinal heparin, which contains Ser as a predominant amino acid, was used for isolation of the carbohydrate-protein linkage region of heparin. Two glycoserines were isolated in a molar ratio of 96:4 after an exhaustive digestion with a mixture of bacterial heparinase and heparitinases. Their structures were determined by composition analysis, heparitinase digestion, co-chromatography with an authentic glycoserine on high performance liquid chromatography, and by 500-MHz one- and two-dimensional 1H NMR spectroscopy. The structure of the major one is delta GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and that of the minor is delta GlcA beta 1-4GlcNAc(6-O-sulfate) alpha 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser. The novel 6-O-sulfated GlcNAc residue was demonstrated to occur in the vicinity of the carbohydrate-protein linkage region. The Gal residues were nonsulfated, in contrast to the sulfated Gal structures recently discovered in the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans. The structural features are discussed in relation to biosynthetic mechanisms of the heparin glycosaminoglycans.     

Structural determination of novel tetra- and hexasaccharide sequences isolated from chondroitin sulfate H (oversulfated dermatan sulfate) of hagfish notochord

Oversulfated chondroitin sulfate H (CS-H) isolated from hagfish notochord is a unique dermatan sulfate consisting mainly of IdoAalpha1-3GalNAc(4S,6S), where IdoA, GalNAc, 4S and 6S represent L-iduronic acid, Nacetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. Several tetra- and hexasccharide fractions were isolated from CS-H after partial digestion with bacterial chondroitinase B to investigate the sequential arrangement of the IdoAalpha1-3GalNAc(4S,6S) unit in the CS-H polysaccharide. A structural analysis of the isolated oligosaccharides by enzymatic digestions, mass spectrometry and 1H NMR spectroscopy demonstrated that the major tetrasaccharides shared the common disulfated core structure delta4,5HexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc (4S) with 0 approximately 3 additional O-sulfate groups, where delta4,5HexA represents 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid. The major hexasaccharides shared the common trisulfated core structure delta4,5HexAalpha1-3 GalNAc(4S)beta1-4 IdoAalpha1-3 GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc(4S) with 1 approximately 4 additional O-sulfate groups. Some extra sulfate groups in both tetra- and hexasaccharides were located at the C-2 position of a delta4,5HexA or an internal IdoA residue, or C-6 position of 4-O-sulfated GalNAc residues, forming the unique disulfated or trisulfated disaccharide units, IdoA (2S)-GalNAc(4S), IdoA-GalNAc(4S,6S) and IdoA (2S)-GalNAc(4S,6S), where 2S represents 2-O-sulfate. Of the demonstrated sequences, five tetra- and four hexasaccharide sequences containing these units were novel.     

Isolation and structural determination of novel sulfated hexasaccharides from squid cartilage chondroitin sulfate E that exhibits neuroregulatory activities

Squid cartilage chondroitin sulfate E (CS-E) exhibits various biological activities, including anticoagulant activities, lymphoid regulatory activities, and neuroregulatory activities [Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413]. These activities are expressed through molecular interactions with specific proteins, including heparin cofactor II, selectins, CD44, chemokines, and the heparin-binding growth factor midkine. Hence, the sugar sequence information is essential for a better understanding of the CS-E functions. Previously, several novel tetrasaccharides containing the unreported 3-O-sulfated glucuronic acid (GlcA) were isolated after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, hexasaccharides were isolated to obtain more detailed sequence information, especially around the GlcA(3-O-sulfate) residue, and were characterized by fast atom bombardment mass spectrometry and 500 or 600 MHz (1)H NMR spectroscopy. The findings demonstrate one tetrasulfated and five pentasulfated hexasaccharide sequences, five of them being novel. They were composed of three disaccharide building units of either A [GlcA(beta1-3)GalNAc(4-O-sulfate)], E [GlcA(beta1-3)GalNAc(4,6-O-disulfate)], K [GlcA(3-O-sulfate)(beta1-3)GalNAc(4-O-sulfate)], L [GlcA(3-O-sulfate)(beta1-3)GalNAc(6-O-sulfate)], or M [GlcA(3-O-sulfate)(beta1-3)GalNAc(4,6-O-disulfate)], forming E-A-A, M-A-A, K-L-A, E-E-A, K-K-A, and A-M-A hexasaccharide sequences. The K-L tetrasaccharide sequence is to date unreported. The isolated sequences appear to indicate the occurrence of an unreported GlcA 3-O-sulfotransferase specific for chondroitin sulfate. The obtained sequence information will be useful for investigating the structure-function relationship and biosynthesis of CS-E.     

Glycosaminoglycans from marine sources as therapeutic agents

Glycosaminoglycans (GAGs) in marine animals are different to those of terrestrial organisms, mainly in terms of molecular weight and sulfation. The therapeutic properties of GAGs are related to their ability to interact with proteins, which is very much influenced by sulfation position and patterns. Since currently GAGs cannot be chemically synthesized, they are sourced from natural products, with high intra- but also inter-species variability, in terms of chain length, disaccharide composition and sulfation pattern. Consequently, sulfated GAGs are the most interesting molecules in the marine environment and constitute the focus of the present review. In particular, chondroitin sulfate (CS) appears as the most promising compound. CS-E chains [GlcA-GalNAc(4S,6S)] extracted from squid possess antiviral and anti-metastatic activities and seem to impart signalling properties and improve the mechanical performance of cartilage engineering constructs; Squid CS-E and octopus CS-K [GlcA(3S)-GalNAc(4S)], dermatan sulfate (DS) from sea squirts [-iK units, IdoA(3S)-GalNAc(4S)] and sea urchins [-iE units, IdoA-GalNAc(4S,6S)] and hybrids CS/DS from sharks (-B/iB [GlcA/IdoA(2S)-GalNAc(4S)], -D/iD [GlcA/IdoA(2S)-GalNAc(6S)] and –E/iE units [GlcA/IdoA-GalNAc(4S,6S)]) promote neurite outgrowth and could be valuable materials for nerve regeneration. Also displaying antiviral and anti-metastatic properties, a rare CS with fucosylated branches isolated from sea cucumbers is an anticoagulant and anti-inflammatory agent. In this same line, marine heparin extracted from shrimp and sea squirt has proven anti-inflammatory properties, with the added advantage of decreased risk of bleeding because of its low anticoagulant activity.     

Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate

A differentiated population of cells with metachromatically staining granules and surface IgE receptors was obtained from mouse bone marrow cultured for 2 weeks in the presence of conditioned medium derived from concanavalin A-stimulated splenocytes. The cells were found to incorporate large amounts of [35S]sulfate into an intracellular 35S-labeled proteoglycan of Mr approximately 200,000 containing a maximum of seven glycosaminoglycan side chains (Mr = 25,000). After chondroitinase ABC treatment of density gradient-purified [3H] serine-labeled proteoglycan, the resulting core was Mr approximately 26,000 as assessed by gel filtration. Two-dimensional cellulose acetate electrophoresis of beta-eliminated 35S-labeled glycosaminoglycan revealed a single type of glycosaminoglycan that migrated at the position of oversulfated chondroitin sulfate E from squid cartilage. Chondroitinase ABC degradation of the 35S-labeled glycosaminoglycan yielded two cleavage products in approximately equal molar amounts which co-migrated in both descending paper chromatography and high voltage paper electrophoresis with a monosulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and a disulfated disaccharide, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-6-di-O-sulfo-D-galactose. The release of some free [35S]sulfate from the oversulfated disaccharide with either chondro-4-sulfatase or chondro-6-sulfatase and the complete desulfation by their combined action established that the oversulfated disaccharide contained N-acetylgalactosamine-4,6-disulfate. The 35S]labeled proteoglycan of these unique IgE receptor-bearing and histamine-containing cells, therefore, is composed of chondroitin sulfate E rather than heparin glycosaminoglycan, and thus is the first identification of such an intracellular localized proteoglycan in a mammalian cell.     

Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111

We synthesized the biotinylated chondroitin sulfate tetrasaccharides CS-CC [-3)βGalNAc6S(1–4)βGlcA(1-]₂ and CS-DD [-3)βGalNAc6S(1–4)βGlcA2S(1-]₂ which possess sulfate groups at O-6 of GalNAc and an additional sulfate group at O-2 of GlcA, respectively. We also analyzed interactions among CS-CC and CS-DD and the antibodies 2H6 and LY111, both of which are known to bind with CS-A, while CS-DD was shown for the first time to bind with both antibodies.     

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: DeltaUA(beta1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(beta1-4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: DeltaUA(beta1-3)GalNAc4S(beta1-4)GlcA-ol and DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S-ol and GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.     

The phosphorylated and/or sulfated structure of the carbohydrate-protein-linkage region isolated from chondroitin sulfate in the hybrid proteoglycans of Engelbreth-Holm-Swarm mouse tumor.

The structure of the linkage region of chondroitin sulfate chains attached to the hybrid proteoglycans of the Engelbreth-Holm-Swarm mouse tumor was investigated. The peptidoglycan fraction which contains oversulfated chondroitin sulfate rich in the GlcA beta 1-3GalNAc-4,6-diO-sulfate unit and undersulfated heparan sulfate rich in GlcA beta 1-4GlcNAc and GlcA beta 1-4GlcN-2N-sulfate units was isolated after exhaustive protease digestion of the acetone powder of the tumor tissue, (GlcA, glucuronic acid; GalNAc, 2-deoxy-2-N-acetylamino-D-galactose). Glycosaminoglycans were released by beta-elimination using NaB3H4 and digested with chondroitinase ABC. The linkage region fraction was separated from heparan sulfate by gel filtration and fractionated by HPLC on an amine-bound silica column. Six radiolabeled compounds (L1-L6) were obtained and structurally analyzed by cochromatography with authentic hexasaccharide alditols recently isolated by us from the linkage region, and by digestion using chondroitinase ACII, alkaline phosphatase and beta-galactosidase in conjugation with HPLC. These compounds shared the conventional hexasaccharide backbone structure: delta GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol, (delta GlcA, delta 4.5-GlcA or D-gluco-4-enepyranosyluronic acid). L1 was not sulfated or phosphorylated. L2 and L4 were monosulfated at C-6 and C-4 of the GalNAc residue, respectively. Upon alkaline phosphatase digestion, L3, L5 and L6 were converted to L1, L2 and L4, respectively. Analysis of the periodate oxidation products indicated that the phosphate group in L3, L5 and L6 is located at C-2 of Xyl-ol. These results suggest that Xyl-2-O-phosphate is associated with both 4-O-sulfated and 6-O-sulfated GalNAc units and does not directly determine the sulfation pattern of chondroitin sulfate.     

A method for the sequence analysis of dermatan sulphate.

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.     

Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.     

Structural elucidation of glycosaminoglycans through characterization of disaccharides obtained after fragmentation by hydrazine-nitrous acid treatment

Hydrazinolysis of glycosaminoglycans to bring about N-deacetylation followed by nitrous acid treatment to effect deaminative cleavage at alternating hexosamine residues has been used to make possible identification and quantitation of disaccharide sequences and position of O-sulfate substitution in nanogram amounts of these polymers. After radiolabeling by NaB3H4 reduction the hydrazine-nitrous acid products were fractionated on Dowex 1 and further resolved by thin-layer chromatography into disaccharides terminating in either sulfated or unsulfated anhydromannitol or anhydrotalitol. Fragmentation of hyaluronic acid, keratan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and heparin yielded a total of 14 disaccharides comprising the major sequences (greater than 1 mol%) occurring in mammalian glycosaminoglycans. Disaccharides representing the predominant variants of the chondroitin sulfates [GlcUA beta 1----3anhydrotalitol(4-SO4) and GlcUA beta 1----3anhydrotalitol(6-SO4)] as well as of dermatan sulfate chains [IdUA alpha 1----3anhydrotalitol(4-SO4) and GlcUA beta 1----3anhydrotalitol(4-SO4)] chains could readily be quantitated by this approach. In the case of heparin a comparison of the disaccharides produced by direct nitrous acid and hydrazine-nitrous acid treatments moreover provided an assessment of the distribution of N-sulfate groups. The characterization of the various disaccharides by Smith periodic acid degradation and glycosidase digestions was facilitated by the preparation and thin-layer chromatographic resolution of the complete series of monosulfated derivatives of anhydromannitol and anhydrotalitol; the sulfate esters were shown to be stable to both the hydrazine and nitrous acid treatments. The high sensitivity of the hydrazine-nitrous acid fragmentation procedure should prove useful in the structural elucidation of cell surface and basement membrane proteoglycans as well as other sulfated glycoconjugates which are present in small amounts.     

Biosynthesis of heparin. Enzymatic sulfation of pentasaccharides

Heparin-derived pentasaccharides with the general structures GlcN-GlcA/IdoA-GlcN-GlcA/IdoA-GlcN (where GlcA represents D-glucuronic acid and IdoA represents L-iduronic acid) and GlcNSO3-GlcA/IdoA-GlcNSO3-GlcA/IdoA- GlcNSO3 (where -NSO3 represents an N-sulfate group) were tested as exogenous sulfate acceptors in incubations with adenosine 3'-phosphate 5'-[35S]phosphosulfate and microsomal enzymes from a heparin-producing mouse mastocytoma. No transfer occurred to the N-unsubstituted pentasaccharide containing only L-iduronic acid, but the other three isomers incorporated various amounts of 35S, which was totally present in N-sulfate groups. After complete chemical N-sulfation, all four pentasaccharides served as acceptors in O-sulfotransferase reactions and incorporated from 20 to greater than 200 times as much radioactivity as did the nonsulfated parent compounds. The C-6 position of the internal glucosamine unit was labeled preferentially, irrespective of the structures of the adjacent hexuronic acid units. Significant 2-O-35S-sulfation of IdoA units occurred in both -IdoA-Glc-NSO3-GlcA- and -GlcA-GlcNSO3-IdoA- sequences, whereas no significant sulfation of GlcA residues was detected. The pentasaccharide GlcNSO3-GlcA-Glc-NSO3-GlcA-GlcNSO3 thus can be used as a selective substrate in assays for glucosaminyl-6-O-sulfotransferase activity. The antithrombin-binding region, essential for the blood anticoagulant activity of heparin, has been identified as a pentasaccharide sequence with the predominant structure GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-++ +IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R represents either a sulfate or an acetyl group and -OSO3 represents an O-sulfate/ester sulfate group, with locations of O-sulfate groups indicated in parentheses) (Lindahl U., Thunberg, L., B?ckstr?m, G., Riesenfeld, J., Nordling, K., and Bj?rk, I. (1984) J. Biol. Chem. 259, 12368-12376). The products of [35S]sulfate transfer to the pentasaccharide GlcNSO3-GlcA-GlcNSO3-IdoA-GlcNSO3 contained molecules with high affinity for antithrombin, corresponding to 0.3-0.5% of the total label. Structural analysis suggested the occurrence of O-[35S]sulfate groups at both C-6 of the nonreducing terminal glucosamine unit and C-3 of the internal glucosamine unit. No products with high affinity for antithrombin were formed from the pentasaccharides that had a different monosaccharide sequence than the binding region; and moreover, these oligosaccharides appeared unable to incorporate glucosaminyl 3-O-sulfate groups. These findings point to the importance of the uronic acid sequence in the generation of the antithrombin-binding region of heparin.     

Selective depolymerisation of dermatan sulfate: production of radiolabelled substrates for alpha-L-iduronidase, sulfoiduronate sulfatase, and beta-D-glucuronidase

Radiolabelled disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and sulfoiduronate sulfatase have been prepared from dermatan sulfate by application in sequence of N-deacetylation, deaminative cleavage, and reduction with NaBT4. The yield of disaccharides was approximately 87% of the total oligosaccharide fraction. Five disaccharides were isolated and tentatively identified. The major disaccharide, O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA-anT4S), represented approximately 75% of the total disaccharide fraction. The other disaccharides were O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA2S-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (GlcA-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 6-sulfate (GlcA-anT6S), and O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol (IdoA-anT), which represented approximately 4.5, 11.2, 1.0, and 1.8%, respectively, of the total disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls, IdoA-anT4S was shown to be a sensitive substrate for alpha-L-iduronidase to produce 2,5-anhydro-D-talitol 4-sulfate (anT4S). Activity toward IdoA-anT4S was not observed with fibroblast homogenates from alpha-L-iduronidase-deficient patients (Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from beta-D-glucuronidase-deficient patients (Mucopolysaccharidosis Type VII). IdoA-anT4S was hydrolysed by alpha-L-iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro-D-[1-3H]talitol residues is an important structural determinant in the mechanism of action of alpha-L-iduronidase on disaccharide substrates. IdoA2S-anT4S was degraded to IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from alpha-L-iduronidase-deficient and sulfoiduronate sulfatase-deficient (Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and IdoA-anT4S (and anT4S), respectively.     

Two N-acetylgalactosaminyltransferase are involved in the biosynthesis of chondroitin sulfate

Two N-acetylgalactosaminyltransferases, designated I and II, have been purified from the microsomal fraction of calf arterial tissue and separated on Bio-Gel A. N-Acetylgalactosaminyltransferase I was purified 450-fold. It requires Mn2+ for maximal activity and transfers N-acetylgalactosamine residues from UDP-[1-3H]GalNAc in beta-glycosidic configuration to the non-reducing terminus of the acceptor substrates GlcA(beta 1-3)Gal(beta 1-3)Gal, GlcA(beta 1-3)Gal(beta 1-4)Glc and GlcA(beta 1-3)Gal. Even-numbered chondroitin oligosaccharides serve as acceptors for N-acetylgalactosaminyltransferase II, which transfers N-acetylgalactosamine from UDP-[1-3H]GalNAc to the non-reducing glucuronic acid residues of oligosaccharide acceptor substrates. Maximum transfer rates were obtained with a decasaccharide derived from chondroitin. Longer or shorter-chain chondroitin oligosaccharides are less effective acceptor substrates. All reaction products formed by N-acetylgalactosaminyltransferases I and II are substrates of beta-N-acetylhexosaminidase, which splits off the transferred [1-3H]GalNAc completely. In the microsomal fraction N-acetylgalactosaminyltransferase II had a 300-fold higher specific activity than N-acetylgalactosaminyltransferase I. In contrast to enzyme I, enzyme II loses much of its activity during the purification procedure and undergoes rapid thermodenaturation. GlcA-Gal-Gal is a characteristic sequence of the carbohydrate-protein linkage region of proteochondrioitin sulfate. The acceptor capacity of this trisaccharide suggests that N-acetylgalactosaminyltransferase I is involved in the synthesis of the carbohydrate-protein linkage region. Since N-acetylgalactosaminyltransferase II is highly specific for chondroitin oligosaccharides, we conclude that it participates in chain elongation during chondroitin sulfate synthesis.