Structural diversity of N-sulfated heparan sulfate domains: distinct modes of glucuronyl C5 epimerization, iduronic acid 2-O-sulfation, and glucosamine 6-O-sulfation

The N-sulfated regions (NS domains) represent the modified sequences of heparan sulfate chains and mediate interactions of the polysaccharide with proteins. We have investigated the relationship between the type/extent of polymer modification and the length of NS domains in heparan sulfate species from human aorta, bovine kidney, and cultured NMuMG and MDCK cells. C5 epimerization of D-glucuronic acid to L-iduronic acid was found to be extensive and essentially similar in all heparan sulfate species studied, regardless of domain size, whereas the subsequent 2-O-sulfation of the formed iduronic acid residues varies appreciably. In aorta heparan sulfate, up to 90% of the formed iduronate residues were 2-O-sulfated, whereas in kidney heparan sulfate 2-O-sulfation occurred only in 相似文献   

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.     

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.     

Common binding sites for beta-amyloid fibrils and fibroblast growth factor-2 in heparan sulfate from human cerebral cortex.

Heparan sulfate found in the cerebral plaques of Alzheimer's disease binds to beta-amyloid (Abeta) fibrils. This interaction has been proposed to enhance fibril deposition and mediate Abeta-induced glia activation and neurotoxicity. On the other hand, heparan sulfate augments signaling of fibroblast growth factor-2 (FGF-2), a neuroprotective factor that antagonizes the neurotoxic effects of Abeta. We defined structures in heparan sulfate from human cerebral cortex that bind Abeta fibrils. The minimal binding site is found in N-sulfated hexasaccharide domains and contains critical 2-O-sulfated iduronic acid residues. By contrast, binding of Abeta monomers requires, in addition, 6-O-sulfate groups on glucosamine residues. The binding specificity of fibrillar Abeta is shared by FGF-2, and we here show that cerebral heparan sulfate domains selected for binding to Abeta-(1-40) fibrils bind also to FGF-2. These data suggest that neurotoxic and neuroprotective signals may converge by competing for the same binding sites on the heparan sulfate chain.     

Coupling of N-deacetylation and N-sulfation in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase

The coordination of N-deacetylation and N-sulfation of heparan sulfate was examined in wild-type Chinese hamster ovary cells and mutant pgsE-606. This mutant expresses about 3-fold less N-sulfotransferase activity, which causes the proportion of N-sulfated GlcN residues in heparan sulfate to decline from 39 to 21% of total GlcN (Bame, K.J., and Esko, J.D. (1989) J. Biol. Chem. 264, 8059-8065). In this report, we show that microsomes from pgsE-606 cells have about twice the N-deacetylase activity found in microsomes from wild-type cells. However, N-deacetylation in vivo was actually depressed since heparan sulfate preparations from the mutant contained very few unsubstituted GlcN residues and 2-fold less N-sulfated GlcN residues. Treatment of mutant cells with chlorate, a general inhibitor of sulfation, depressed adenosine 3'-phosphate-5'-phosphosulfate pools more than 10-fold and further reduced the extent of N-sulfation from 21% to less than 6% of total GlcN. Unsubstituted GlcN residues accumulated under these conditions to the extent that N-sulfated residues declined. Thus, N-deacetylation remained depressed in the mutant in the presence of chlorate. These findings show that N-deacetylation is regulated in vivo and support the idea that the activity of N-deacetylase may be linked to N-sulfotransferase.     

Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O- sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.      

Structural characteristics of heparan sulfates with varying sulfate contents.

Structural properties of heparan sulfate preparations from hog mucosa and beef lung sources were obtained by application of Smith degradation and nitrous acid reactions. Products formed by these reactions indicated that most of the iduronic acid present in these mucopolysaccharides is ester sulfated, whereas N-sulfated glucosamine residues are ester sulfated much less frequently. Repeating units with sulfated iduronic acid found to occur almost entirely in single sequences. Futhermore, the iduronic acid moieties may be bound to either N-acetylated or N-sulfated glucosamine units, with these occuring at either end of the uronic acid unit.     

Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3-O-sulfotransferase 5

The 3-O-sulfation of glucosamine is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). Both heparan sulfate 3-O-sulfotransferase -1 (3-OST-1) and 3-O-sulfotransferase-5 (3-OST-5) transfer sulfate to the 3-OH group of glucosamine to generate antithrombin-binding heparan sulfate (HS(act)). Here, we reported the isolation and characterization of the antithrombin-binding HS oligosaccharides generated by 3-OST-5 (3-OST-5 oligo(act)). (3)H-labeled HS of Chinese hamster ovary cells was exhaustively modified by 3-OST-1 to remove the 3-OST-1 modification sites followed by antithrombin-affinity fractionation. The non-antithrombin-binding fraction of 3-OST-1 pretreated HS was further modified by 3-OST-5 to generate additional antithrombin-binding HS, which was designated as 3-OST-5 HS(act). Structural analysis of 3-OST-5 HS(act) revealed that the antithrombin-binding site of 3-OST-5 HS(act) is located within a domain clustered with N-sulfated glucosamine units. We also isolated 3-OST-5 antithrombin-binding oligosaccharides (3-OST-5 oligo(act)) from high pH nitrous acid degraded 3-OST-5 HS(act). A disaccharide analysis revealed that 3-OST-5 oligo(act) were composed of multiple 3-O-sulfated glucosamine units. Our results provide additional insights on the relationship between the anticoagulant activity and structure of HS.     

Detection of 2-O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of alpha-d-N-acetylglucosaminyl-2-O-sulfo-alpha-l-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V(H)3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.     

Undersulfated heparan sulfate in a Chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase

Heparan sulfate N-sulfotransferase catalyzes the transfer of sulfate groups from adenosine 3'-phosphate, 5'-phosphosulfate to the free amino groups of glucosamine residues in heparan sulfate. We have identified a Chinese hamster ovary cell mutant, designated pgsE-606, which is 3-5-fold defective in N-sulfotransferase activity. The residual enzyme activity is indistinguishable from the wild-type enzyme with respect to Km values for adenosine 3'-phosphate,5'-phosphosulfate and N-desulfoheparin, pH dependence, Arrhenius activation energy, and thermal lability. The mutation is recessive, and mixing experiments indicate that the mutant does not produce soluble antagonists of N-sulfotransferase. Inspection of the heparan sulfate chains from the mutant showed that the extent of N-sulfation is reduced about 2-3-fold. The addition of sulfate to hydroxyl groups on the chain is reduced to a similar extent, suggesting that N-sulfation and O-sulfation are normally coupled. Nitrous acid fragmentation of the chains showed that N-sulfated glucosamine residues are spaced much less frequently than in heparan sulfate from wild-type cells. The close correlation of enzyme activity to the number and position of N-sulfate groups indicates that N-sulfotransferase plays a pivotal role in determining the extent of sulfation of heparan sulfate.     

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.     

The antiproliferative activity of arterial heparan sulfate resides in domains enriched with 2-O-sulfated uronic acid residues.

Heparan sulfate isolated from bovine arterial tissue by a multistep purification procedure or from arterial tissue proteoheparan sulfate by beta-elimination exhibits antiproliferative activity toward arterial smooth muscle cells when added to subconfluent cell cultures in a concentration of 50-100 micrograms/ml medium. Enzymatic disintegration of heparan sulfate by heparitinases I and II and isolation of the resulting oligosaccharides indicate that the antiproliferative activity of the heparan sulfate molecule resides in a sulfate-rich octa/decasaccharide domain which is separated by longer sequences of sulfate-free or sulfate-poor N-acetylglucosamine containing disaccharide units. The octa/decasaccharide fraction has a 3-4-fold higher antiproliferative activity than the native heparan sulfate molecule and contains 45% of a disulfated disaccharide which consists of 2-O-sulfated uronic acid and N-sulfated glucosamine (UA(2S)-GlcNS and 12% of a trisulfated disaccharide (UA(2S)-GlcNS(6S). A sulfate-rich hexasaccharide fraction containing 14% of the disulfated disaccharide but 18% of the trisulfated disaccharide has negligible antiproliferative activity. The results indicate the presence of specific structural determinants in the arterial heparan sulfate molecule which may have the function of an endogenous inhibitor of arterial smooth muscle cell growth.     

Location of N-unsubstituted glucosamine residues in heparan sulfate

Functional properties of heparan sulfate (HS) are generally ascribed to the sulfation pattern of the polysaccharide. However, recently reported functional implications of rare N-unsubstituted glucosamine (GlcNH(2)) residues in native HS prompted our structural characterization of sequences around such residues. HS preparations were cleaved with nitrous acid at either N-sulfated or N-unsubstituted glucosamine units followed by reduction with NaB(3)H(4). The labeled products were characterized following complementary deamination steps. The proportion of GlcNH(2) units varied from 0.7-4% of total glucosamine in different HS preparations. The GlcNH(2) units occurred largely clustered at the polysaccharide-protein linkage region in intestinal HS, also more peripherally in aortic HS. They were preferentially located within N-acetylated domains, or in transition sequences between N-acetylated and N-sulfated domains, only 20-30% of the adjacent upstream and downstream disaccharide units being N-sulfated. The nearest downstream (toward the polysaccharide-protein linkage) hexuronic acid was invariably GlcUA, whereas the upstream neighbor could be either GlcUA or IdoUA. The highly sulfated but N-unsubstituted disaccharide unit, -IdoUA2S-GlcNH(2)6S-, was detected in human renal and porcine intestinal HS, but not in HS from human aorta. These results are interpreted in terms of a biosynthetic mechanism, whereby GlcNH(2) residues are formed through regulated, incomplete action of an N-deacetylase/N-sulfotransferase enzyme.     

Irreversible glucuronyl C5-epimerization in the biosynthesis of heparan sulfate

Glucuronyl C5-epimerase catalyzes the conversion of d-glucuronic acid to l-iduronic acid units in heparan sulfate biosynthesis. Substrate recognition depends on the N-substituent pattern of the heparan sulfate precursor polysaccharide and requires the adjacent glucosamine residue toward the non-reducing end to be N-sulfated. Epimerization of an appropriately N-sulfated substrate is freely reversible in a soluble system, with equilibrium favoring retention of d-gluco configuration (Hagner-McWhirter, A., Lindahl, U., and Li, J.-P. (2000) Biochem. J. 347, 69-75). We studied the reversibility of the epimerase reaction in a cellular system, by incubating human embryonic kidney 293 cells with d-[5-(3)H]galactose. The label was incorporated with glucuronic acid units into the heparan sulfate precursor polysaccharide and was lost upon subsequent C5-epimerization to iduronic acid. However, analysis of oligosaccharides obtained by deaminative cleavage of the mature heparan sulfate chains indicated that all glucuronic acid units retained their C5-(3)H label, irrespective of whether they had occurred in sequences susceptible or resistant to the epimerase. All (3)H-labels of the final products resisted incubation with epimerase in a soluble system, apparently due to blocking O-sulfate groups. These results indicate that glucuronic acid C5-epimerization is effectively irreversible in vivo and argue for a stringent organization of the biosynthetic machinery.     

Oligosaccharide library-based assessment of heparan sulfate 6-O-sulfotransferase substrate specificity

Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.     

Hexuronyl C5-epimerases in alginate and glycosaminoglycan biosynthesis

The sugar residues in most polysaccharides are incorporated as their corresponding monomers during polymerization. Here we summarize the three known exceptions to this rule, involving the biosynthesis of alginate, and the glycosaminoglycans, heparin/heparan sulfate and dermatan sulfate. Alginate is synthesized by brown seaweeds and certain bacteria, while glycosaminoglycans are produced by most animal species. In all cases one of the incorporated sugar monomers are being C5-epimerized at the polymer level, from D-mannuronic acid to L-guluronic acid in alginate, and from D-glucuronic acid to L-iduronic acid in glycosaminoglycans. Alginate epimerization modulates the mechanical properties of seaweed tissues, whereas in bacteria it seems to serve a wide range of purposes. The conformational flexibility of iduronic acid units in glycosaminoglycans promotes apposition to, and thus functional interactions with a variety of proteins at cell surfaces and in the extracellular matrix. In the bacterium Azotobacter vinelandii the alginates are being epimerized at the cell surface or in the extracellular environment by a family of evolutionary strongly related modular type and Ca(2+)-dependent epimerases (AlgE1-7). Each of these enzymes introduces a specific distribution pattern of guluronic acid residues along the polymer chains, explaining the wide structural variability observed in alginates isolated from nature. Glycosaminoglycans are synthesized in the Golgi system, through a series of reactions that include the C5-epimerization reaction along with extensive sulfation of the polymers. The single, Ca(2+)-independent, epimerase in heparin/heparan sulfate biosynthesis and the Ca(2+)-dependent dermatan sulfate epimerase(s) also generate variable epimerization patterns, depending on other polymer-modification reactions. The alginate and heparin epimerases appear unrelated at the amino acid sequence level, and have probably evolved through independent evolutionary pathways; however, hydrophobic cluster analysis indicates limited similarity. Seaweed alginates are widely used in industry, while heparin is well established in the clinic as an anticoagulant.     

The disaccharide composition of heparins and heparan sulfates

Heparin and heparan sulfate can be cleaved selectively at their N-sulfated glucosamine residues by direct treatment with nitrous acid at pH 1.5. These polymers can also be cleaved selectively at their N-acetylated glucosamine residues by first N-deacetylating with hydrazine and then treating the products with nitrous acid at pH 4. These procedures have been combined and optimized for the conversion of these glycosaminoglycan chains into their disaccharide units. A modified hydrazinolysis procedure in which the glycosaminoglycans were heated with hydrazine:water (70:30) containing 1% hydrazine sulfate gave rapid rates of N-deacetylation and minimal conversion of the uronic acid residues to their hydrazide derivatives. Under these conditions, N-deacetylation was complete in 4 h and the beta-eliminative cleavage of the polymer chains that occurs during hydrazinolysis (P. N. Shaklee and H. E. Conrad (1984) Biochem. J. 217, 187-197) was eliminated. Treatment of the N-deacetylated polymer with nitrous acid at pH 3 for 15 h at 25 degrees C then gave simultaneous cleavage at the N-unsubstituted glucosamine residues and the N-sulfated glucosamine residues. These deamination conditions minimized, but did not eliminate, the side reaction in which nitrous acid-reactive glucosamine residues undergo ring contraction without glucosaminide bond cleavage. Thus, the disaccharides were obtained in a yield of 90% of those originally present in the glycosaminoglycan chains. Since the ring contraction side reaction occurs randomly at the diazotized glucosamine residues, the disaccharides formed in the pH 3 nitrous acid reaction were recovered in proportions equal to those in the original glycosaminoglycan chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.     

Human follicular fluid heparan sulfate contains abundant 3-O-sulfated chains with anticoagulant activity

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.