Biosynthesis of heparin. O-sulfation of the antithrombin-binding region

The antithrombin-binding region in heparin is a pentasaccharide sequence with the predominant structure GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-IdoA -(2-OSO3)-GlcNSO3(6-OSO3) (where GlcA and IdoA represent D-glucuronic and L-iduronic acid, respectively), in which the 3-O-sulfate residue on the internal glucosaminyl unit is a marker group for this particular region of the polysaccharide molecule. A heparin octasaccharide which contained the above pentasaccharide sequence was N/O-desulfated and re-N-sulfated and was then incubated with adenosine 3'-phosphate 5'-phospho[35S]sulfate in the presence of a microsomal fraction from mouse mastocytoma tissue. Fractionation of the resulting 35S-labeled octasaccharide on antithrombin-Sepharose yielded a high affinity fraction that accounted for approximately 2% of the total incorporated label. Structural analysis of this fraction indicated that the internal glucosamine unit of the pentasaccharide sequence was 3-O-35S-sulfated, whereas both adjacent glucosamine units carried 6-O-[35S]sulfate groups. In contrast, the fractions with low affinity for antithrombin (approximately 98% of incorporated 35S) showed no consistent O-35S sulfation pattern and essentially lacked glucosaminyl 3-O-[35S]sulfate groups. It is suggested that the 3-O-sulfation reaction concludes the formation of the antithrombin-binding region. This proposal was corroborated in a similar experiment using a synthetic pentasaccharide with the structure GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-Id oA (2-OSO3)-GlcNSO3(6-OSO3) as sulfate acceptor. This molecule corresponds to a functional antithrombin-binding region but for the lack of a 3-O-sulfate group at the internal glucosamine unit. The 35S-labeled pentasaccharide recovered after incubation bound with high affinity to antithrombin-Sepharose and contained a 3-O-[35S]sulfate group at the internal glucosamine residue as the only detectable labeled component. The use of this pentasaccharide substrate along with the affinity matrix provides a highly specific assay for the 3-O-sulfotransferase.     

Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate.

The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF.     

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.     

Location of the antithrombin-binding sequence in the heparin chain

The antithrombin-binding region of heparin is a pentasaccharide sequence with the predominant structure -GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-Ido A(2-OSO3)- GlcNSO3(6-OSO3)-. By using the 3-O-sulfated glucosamine residue as a marker for the anti-thrombin-binding sequence, the location of this sequence within the heparin chain was investigated. Heparin with high affinity for antithrombin (HA-heparin) contains few N-acetyl groups located outside the antithrombin-binding region, and cleavage at such groups was therefore expected to be essentially restricted to this region. HA-heparin was cleaved at N-acetylated glucosamine units by partial deacetylation followed by treatment with nitrous acid at pH 3.9, and the resulting fragments with low affinity for anti-thrombin (LA-fragments) were recovered after affinity chromatography on immobilized antithrombin. The LA-fragments were further divided into subfractions of different molecular size by gel chromatography and were then analyzed with regard to the occurrence of the nonreducing terminal GlcA-GlcNSO3(3,6-di-OS-O3)- sequence. Such units were present in small, intermediate-sized as well as large fragments, suggesting that the antithrombin-binding regions were randomly distributed along the heparin chains. In another set of experiments, HA-heparin was subjected to limited, random depolymerization by nitrous acid (pH 1.5), and the resulting reducing terminal anhydromannose residues were labeled by treatment with NaB3H4. The molecular weight distributions of such labeled LA-fragments, determined by gel chromatography, again conformed to a random distribution of the antithrombin-binding sequence within the heparin chains. These results are in apparent disagreement with previous reports (Radoff, S., and Danishefsky, I. (1984) J. Biol. Chem. 259, 166-172; Rosenfeld, L., and Danishefsky, I. (1988) J. Biol. Chem. 263, 262-266) which suggest that the antithrombin-binding region is preferentially located at the nonreducing terminus of the heparin molecule.     

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.     

A sulfatase specific for glucuronic acid 2-sulfate residues in glycosaminoglycans

Although 2-O-sulfated L-iduronic acid (IdoA) residues have been known to occur in heparin, 2-O-sulfated D-glucuronic acid (GlcA) residues have been reported only recently (Bienkowski, M. J., and Conrad, H. E. (1985) J. Biol. Chem. 250, 356-365). Disaccharides prepared by cleavage of heparin and N-deacetylated chondroitin 6-sulfate with nitrous acid were used to demonstrate a new sulfatase that catalyzed the removal of the 2-O-sulfate substituents from GlcA but not IdoA residues. The deamination products were labeled by NaB3H4 reduction to give disaccharides from heparin and chondroitin sulfate which had reducing terminal 2,5-anhydro-D-mannitol ([3H]AManR) and 2,5-anhydro-D-talitol ([3H]ATalR) residues, respectively. IdoA(2-SO4)-[3H]AManR(6-SO4) from heparin and GlcA(2-SO4)-[3H]ATalR(6-SO4) from chondroitin sulfate were purified for use as substrates. GlcA(2-SO4)-[3H]AManR(6-SO4) was prepared by epimerization of IdoA(2-SO4)-[3H]AManR(6-SO4) with hydrazine at 100 degrees C. Lysosomal enzyme preparations from chick embryo chondrocytes and from two normal human fibroblast cell lines catalyzed the removal of the 2-O-SO4 substituent from the uronic acid residues of IdoA(2-SO4)-[3H]AManR(6-SO4), GlcA(2-SO4)-[3H] AManR(6-SO4), and GlcA(2-SO4)-[3H]ATalR(6-SO4). In contrast, a lysosomal enzyme preparation from a human fibroblast cell line deficient in idurono-2-sulfatase (Hunter's-syndrome), which had no activity on the IdoA(2-SO4)-[3H]AManR(6-SO4), converted GlcA(2-SO4)-[3H]AManR(6-SO4) to a mixture of GlcA-[3H] AManR(6-SO4) and [3H]AManR(6-SO4). This enzyme also converted GlcA(2-SO4)-[3H]ATalR(6-SO4) to a mixture of GlcA-[3H]ATalR(6-SO4) and [3H]ATalR(6-SO4). Digestion of both GlcA(2-SO4)-[3H]AManR(6-SO4) and GlcA(2-SO4)-[3H]ATalR(6-SO4) was inhibited by 35SO2-4 and was arrested at the monosulfated disaccharide stage by 1,4-saccharolactone. The glucurono-2-sulfatase exhibited a pH optimum of 4. The results indicate that there exists a separate sulfatase for the removal of sulfate substituents from C-2 of GlcA residues in glycosaminoglycans.     

A synthetic heparan sulfate pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers.

It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.     

Biosynthesis of heparin. Availability of glucosaminyl 3-O-sulfation sites

Heparin preparations isolated from pig intestinal mucosa and from bovine lung were fractionated with regard to affinity for antithrombin. The resulting fractions, with high (HA) or low (LA) affinity for the proteinase inhibitor, were analyzed by 13C NMR or by identification of di- and tetrasaccharides obtained through deaminative cleavage with nitrous acid. Structural differences between corresponding HA and LA fractions were essentially restricted to minor constituents, in particular 3-O-sulfated glucosamine units that occurred (1 or 2 residues/chain) in all HA preparations but were scarce or absent in LA heparin. The HA fractions also consistently showed higher contents of nonsulfated iduronic acid and, to a lesser extent, N-acetylated glucosamine units than the LA fractions. The two tetrasaccharide sequences, -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3- and -IdoA-GlcNAc(6-OSO3)-GlcA-GlcNSO3(6-OSO3)- , recently implicated as part of the acceptor site for glucosaminyl 3-O-sulfate groups (Kusche, M., B?ckstr?m, G., Riesenfeld, J., Petitou, M., Choay, J., and Lindahl, U. (1988) J. Biol. Chem. 263, 15474-15484), were identified in mucosal LA heparin; it was calculated that the preparation contained approximately one potential acceptor site/polysaccharide chain. Yet this material did not yield any labeled HA components on incubation with adenosine 3'-phosphate 5'-phospho-[35S]sulfate in the presence of glucosaminyl 3-O-sulfotransferase, solubilized from a mouse mastocytoma microsomal fraction. The failure to incorporate any 3-O-sulfate groups could conceivably be explained by the occurrence of a D-glucuronic rather than L-iduronic acid unit linked at the reducing ends of the above tetrasaccharide sequences. Alternatively, 3-O-sulfation may be restricted by other, as yet unidentified, inhibitory structural elements that are preferentially expressed in polysaccharide sequences selected for the generation of LA heparin.     

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.     

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.     

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.     

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and/or phosphate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination, and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols, which are nonsulfated, sulfated, and/or phosphorylated, were obtained from the carbohydrate-protein linkage region. Six compounds, containing 0 or 1 sulfate and/or phosphate residue, represent approximately 40% of the isolated linkage hexasaccharide alditols. They were analyzed by chondroitinase ACII or alkaline phosphatase digestion in conjunction with high performance liquid chromatography, and by 500 MHz one- and two-dimensional 1H NMR spectroscopy. All six compounds have the conventional structure in common. Delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol One compound has no sulfate nor phosphate. Two of the monosulfated compounds have a O-sulfate on C-6 or on C-4 of the GalNAc residue. The third monosulfated compound has a novel O-sulfate on C-6 of the Gal residue attached to xylitol. The two phosphorylated compounds have O-phosphate on C-2 of Xyl-ol, and one of them has in addition sulfate on C-6 of GalNAc.     

Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans

Metabolically 35S- or 3H-labeled heparan sulfate was isolated from murine Reichert's membrane, an extraembryonic basement membrane produced by parietal endoderm cells, and from the basement membrane-producing Engelbreth-Holm-Swarm mouse tumor. The polysaccharides were subjected to structural analysis involving identification of products formed on deamination of the polysaccharides with nitrous acid. The polysaccharide from Reichert's membrane contained N- and O-sulfate groups in approximately equal proportions. It bound almost quantitatively and with high affinity to antithrombin. A high proportion of antithrombin-binding sequence was also indicated by the finding that 3-O-sulfated glucosamine residues accounted for about 10% of the total O-sulfate groups. In contrast, at least 80% of the sulfate residues in the heparan sulfate isolated from the mouse tumor were N-substituents. Only a minor proportion of this polysaccharide bound with high affinity to antithrombin, and no 3-O-sulfated glucosamine residues were detected. These results are discussed in relation to the possible functional role of heparan sulfate in basement membranes.     

Characterization of heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells in culture

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled using [35S]sulfate, [3H]serine, [3H]glucosamine, or [3H]mannose as precursors. A species of heparan sulfate proteoglycan was purified using DEAE-Sephacel chromatography under dissociative conditions in the presence of detergent. The heparan sulfate proteoglycan, which constituted approximately 15% of the 35S-labeled proteoglycans in the culture medium has a similar hydrodynamic size (Kd = 0.62 on Sepharose CL-2B) and buoyant density distribution in CsCl density gradients as the low buoyant density dermatan sulfate proteoglycan synthesized by the same granulosa cells and described in the accompanying report (Yanagishita, M., and Hascall, V. C. (1983) J. Biol. Chem. 258, 12847-12856). The heparan sulfate chains (average Mr = 28,000) have an average of 0.8-0.9 sulfate groups/repeating disaccharide, of which 50% are N-sulfate, 30% are alkaline-labile O-sulfate (presumably on the 6-position of glucosamine residues), and 20% are alkaline-resistant O-sulfate groups. Alkaline borohydride treatment released both N-linked oligosaccharide-peptides containing mannose, glucosamine, and sialic acid, and O-linked oligosaccharides. Trypsin digestion of the proteoglycan generated fragments which contain (a) glycosaminoglycan-peptides with an average of 2 heparan sulfate chains/peptide; (b) clusters of O-linked oligosaccharides on peptides; and (c) N-linked oligosaccharide-peptides, which are as small as single N-linked oligosaccharides. The compositions of the O-linked and N-linked oligosaccharides and the trypsin fragments of this heparan sulfate proteoglycan were very similar to those of the low buoyant density dermatan sulfate proteoglycan synthesized by the same cells.     

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.     

Extension and structural variability of the antithrombin-binding sequence in heparin

Oligosaccharides with different affinities for antithrombin were isolated following partial deaminative cleavage of pig mucosal heparin with nitrous acid. The smallest high-affinity component obtained was previously identified as an octasaccharide with the predominant structure: (Formula: see text). The interaction of this octasaccharide, and of deca- and dodecasaccharides containing the same octasaccharide sequence, with antithrombin was studied by spectroscopic techniques. The near-ultraviolet difference spectra, circular dichroism spectra, and fluorescence enhancements induced by adding these oligosaccharides to antithrombin differed only slightly from the corresponding parameters measured in the presence of undegraded high-affinity heparin. Moreover, the binding constants obtained for the oligosaccharides and for high-affinity heparin were similar (1.0-2.9 X 10(7) M-1 at I = 0.3). In contrast, two hexasaccharides corresponding to units 1-6 and 3-8, respectively, of the above sequence showed about a 1000-fold lower affinity for antithrombin, and also induced considerably different spectral perturbations in antithrombin. Since the 1-6 hexasaccharide contains a reducing-terminal anhydromannose residue instead of the N-sulfated glucosamine unit 6 of the intact sequence, these results strongly support our previous conclusion that the N-sulfate group at position 6 is essential to the interaction with antithrombin. The low affinity of the hexasaccharide 3-8 provides further evidence that a pentasaccharide sequence 2-6 constitutes the actual antithrombin-binding region in the heparin molecule. Structural analysis of the various oligosaccharides revealed natural variants with an N-sulfate group substituted for the N-acetyl group at position 2. The preponderance of N-acetyl over N-sulfate groups at this position may be rationalized in terms of the mechanism of heparin biosynthesis, assuming that the D-gluco configuration of unit 3 is an essential feature of the antithrombin-binding region.     

Characterization of fibroblast growth factor 1 binding heparan sulfate domain.

Fibroblast growth factors FGF-1 and FGF-2 mediate their biological effects via heparan sulfate-dependent interactions with cell surface FGF receptors. While the specific heparan sulfate domain binding to FGF-2 has been elucidated in some detail, limited information has been available concerning heparan sulfate structures involved in the recognition of FGF-1. In the current study we present evidence that the minimal FGF-1 binding heparan sulfate sequence comprises 5-7 monosaccharide units and contains a critical trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. N-Sulfated heparan sulfate decasaccharides depleted of FGF-1 binding domains showed dose-dependent and saturable binding to FGF-2. These data indicate that the FGF-1 binding domain is distinct from the minimal FGF-2 binding site, previously shown to contain an IdoA(2-OSO3) residue but no 6-O-sulfate groups. We further show that the FGF-1 binding heparan sulfate domain is expressed in human aorta heparan sulfate in an age-related manner in contrast to the constitutively expressed FGF-2 binding domain. Reduction of heparan sulfate O-sulfation by chlorate treatment of cells selectively impedes binding to FGF-1. The present data implicate the 6-O-sulfation of IdoA(2-OSO3)-GlcNSO3 units in cellular heparan sulfate in the control of the biological activity of FGF-1.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols were obtained from the carbohydrate-protein linkage region and six of them contain 0 or 1 sulfate and/or 1 phosphate residue (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). The other seven compounds, which represent approximately 60% of the isolated linkage hexasaccharides, were analyzed by chondroitinase ACII digestion in conjunction with high performance liquid chromatography and by 500-MHz one- and two dimensional 1H NMR spectroscopy. All seven compounds have the following conventional structure in common. [formula: see text] Two disulfated compounds have an O-sulfate on C-6 of the Gal-2 residue attached to xylitol in combination with an O-sulfate on C-4 or on C-6 of the GalNAc residue. The third disulfated compound has O-sulfate on C-6 of Gal-2, and also on C-6 of Gal-3. Two of the trisulfated compounds also have O-sulfate on C-6 of both Gal-2 and Gal-3 with in addition sulfate on C-6 or C-4 of GalNAc. The other two trisulfated compounds have O-sulfate on C-6 of Gal-2 and on C-4 of Gal-3 in conjunction with sulfate on C-6 or C-4 of GalNAc.     

Synthesis and interaction with midkine of biotinylated chondroitin sulfate tetrasaccharides

Regiospecifically sulfated chondroitin sulfate repeating tetrasaccharides, CS-OO, GlcAβ-GalNAcβ-GlcAβ-GalNAcβ;CS-EE, GlcAβ-GalNAc(4S6S)β-GlcAβ-GalNAc(4S6S)β; and CS-AA, GlcAβ-GalNAc(4S)β-GlcAβ-GalNAc(4S)β, having biotin linked with a hydrophilic linker at the reducing terminal were synthesized effectively by a coupling of the corresponding disaccharide units and regioselective sulfation. CS-EE showed greater affinity for midkine than CS-AA and CS-OO.