Distinct effects on heparan sulfate structure by different active site mutations in NDST-1

Heparan sulfate polymerization and modification take place in the Golgi compartment. The modification reactions are initiated by glucosaminyl N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme that removes N-acetyl groups from selected N-acetyl-d-glucosamine units followed by N-sulfation of the generated free amino groups. Four isoforms of NDST have been identified. NDST-1 and -2 have a wide and largely overlapping tissue distribution, but it is not known if they can act on the same heparan sulfate chain. We have introduced point mutations into NDST-1 cDNA, which selectively destroy the N-deacetylase or N-sulfotransferase activity of the enzyme [Wei, Z., and Swiedler, S. J. (1999) J. Biol. Chem. 274, 1966-70 and Sueyoshi, T., et al. (1998) FEBS Lett. 433, 211-4]. Stable 293 cell lines expressing the NDST-1 mutants were then generated. Structural analyses of heparan sulfate synthesized by these cells and by cells overexpressing wild-type NDST-1 demonstrate that the N-deacetylation step is not only prerequisite but also rate-limiting, determining the degree of N-sulfation. Transfection of mutant NDST-1 lacking N-deacetylase activity had no effect on heparan sulfate sulfation, while cells expressing wild-type enzyme or NDST-1 lacking N-sulfotransferase activity both resulted in the production of oversulfated heparan sulfate. Since no increase in the amount of N-unsubstituted glucosamine residues was seen after transfection of the mutant lacking N-sulfotransferase activity, the results also suggest that two different enzyme molecules can act on the same glucosamine unit. In addition, we show that oversulfation of heparan sulfate produced by cells tranfected with wild-type NDST-1 or the mutant lacking N-sulfotranferase activity results in decreased sulfation of chondroitin sulfate.     

Antibody-based assay for N-deacetylase activity of heparan sulfate/heparin N-deacetylase/N-sulfotransferase (NDST): novel characteristics of NDST-1 and -2

A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs), which are key enzymes in sulfation of heparan sulfate (HS)/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in HSs by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stably transfected with mouse NDST-1 or -2. We found HSs to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta ( approximately 0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 microM (calculation based on [HexA]) being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 microM, respectively). Comparison of various HS preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (>or=6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated HS, and heparin. Our primary findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, whereas other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.     

Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase/N-sulfotransferase 2

Heparin and heparan sulfate are linear sulfated polysaccharides that exert a multitude of biological functions. Heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase isoform 2 (NDST-2), a key enzyme in the biosynthesis of heparin, contains two distinct activities. This bifunctional enzyme removes the acetyl group from N-acetylated glucosamine (N-deacetylase activity) and transfers a sulfuryl group to the unsubstituted amino position (N-sulfotransferase activity). The N-sulfotransferase activity of NDST has been unambiguously localized to the C-terminal domain of NDST. Here, we report that the N-terminal domain of NDST-2 retains N-deacetylase activity. The N-terminal domain (A66-P604) of human NDST-2, designated as N-deacetylase (NDase), was cloned as a (His)(6)-fusion protein, and protein expression was carried out in Escherichia coli. Heparosan treated with NDase contains N-unsubstituted glucosamine and is highly susceptible to N-sulfation by N-sulfotransferase. Our results conclude that the N-terminal domain of NDST-2 contains functional N-deacetylase activity. This finding helps further elucidate the mechanism of action of heparan sulfate N-deacetylase/N-sulfotransferases and the biosynthesis of heparan sulfate in general.     

Is N-sulfation just a gateway modification during heparan sulfate biosynthesis?

Several biologically important growth factor-heparan sulfate (HS) interactions are regulated by HS sulfation patterns. However, the biogenesis of these combinatorial sulfation patterns is largely unknown. N-Deacetylase/N-sulfotrasferase (NDST) converts N-acetyl-d-glucosamine residues to N-sulfo-d-glucosamine residues. This enzyme is suggested to be a gateway enzyme because N-sulfation dictates the final HS sulfation pattern. It is known that O-sulfation blocks C5-epimerase, which acts immediately after NDST action. However, it is still unknown whether O-sulfation inhibits NDST action in a similar manner. In this article we radically change conventional assumptions regarding HS biosynthesis by providing in vitro evidence that N-sulfation is not necessarily just a gateway modification during HS biosynthesis.     

Overexpression of different isoforms of glucosaminyl N-deacetylase/N-sulfotransferase results in distinct heparan sulfate N-sulfation patterns

Functional interactions of heparan sulfate (HS) with selected proteins depend on distinct saccharide sequences which are generated during biosynthesis of the polysaccharide. Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) catalyze both the N-deacetylation and N-sulfation reactions that initiate the modification of the (GlcNAc-GlcA)(n) polysaccharide backbone. The N-acetyl/N-sulfate exchange is restricted to certain regions of the polysaccharide chains, and only these can be further modified by glucuronyl C5-epimerization and O-sulfation at various positions. To investigate whether NDST isoforms influenced differently the structure of HS, murine NDST-1 was overexpressed in human kidney 293 cells, and the structure of the HS produced was compared to HS from NDST-2 overexpressing cells [Cheung, W. F., Eriksson, I., Kusche-Gullberg M., Lindahl, U., and Kjellén, L. (1996) Biochemistry 35, 5250-5256]. The level of N-sulfation increased from 40% in control cells to 60% and 80%, respectively, in NDST-1 and NDST-2 transfected cells. Interestingly, the increase in N-sulfation was accompanied by an increased chain length, while no effect on IdoA content or O-sulfation was seen. The most extended N-sulfated domains were found in HS synthesized by NDST-2 transfected cells. Since both the N-deacetylase and the N-sulfotransferase activities were lower in these cells than in the NDST-1 overexpressing cells, we conclude that, in addition to the level of enzyme expression, the NDST isoform also is important in determining the N-sulfation pattern in HS.     

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.     

Altered heparan sulfate structure in mice with deleted NDST3 gene function

We report the generation and analysis of mutant mice bearing a targeted disruption of the heparan sulfate (HS)-modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 3 (NDST3). NDST3(-/-) mice develop normally, are fertile, and show only subtle hematological and behavioral abnormalities in agreement with only moderate HS undersulfation. Compound mutant mice made deficient in NDST2;NDST3 activities also develop normally, showing that both isoforms are not essential for development. In contrast, NDST1(-/-);NDST3(-/-) compound mutant embryos display developmental defects caused by severe HS undersulfation, demonstrating NDST3 contribution to HS synthesis in the absence of NDST1. Moreover, analysis of HS composition in dissected NDST3 mutant adult brain revealed regional changes in HS sulfation, indicating restricted NDST3 activity on nascent HS in defined wild-type tissues. Taken together, we show that NDST3 function is not essential for development or adult homeostasis despite contributing to HS synthesis in a region-specific manner and that the loss of NDST3 function is compensated for by the other NDST isoforms to a varying degree.     

Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains

Heparan sulfate (HS) proteoglycans influence embryonic development as well as adult physiology through interactions with various proteins, including growth factors/morphogens and their receptors. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. A key step is the initial generation of N-sulfated domains, primary sites for further polymer modification and ultimately for functional interactions with protein ligands. Such domains, generated through action of a bifunctional GlcNAc N-deacetylase/N-sulfotransferase (NDST) on a [GlcUA-GlcNAc](n) substrate, are of variable size due to regulatory mechanisms that remain poorly understood. We have studied the action of recombinant NDSTs on the [GlcUA-GlcNAc](n) precursor in the presence and absence of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the absence of PAPS, NDST catalyzes limited and seemingly random N-deacetylation of GlcNAc residues. By contrast, access to PAPS shifts the NDST toward generation of extended N-sulfated domains that are formed through coupled N-deacetylation/N-sulfation in an apparent processive mode. Variations in N-substitution pattern could be obtained by varying PAPS concentration or by experimentally segregating the N-deacetylation and N-sulfation steps. We speculate that similar mechanisms may apply also to the regulation of HS biosynthesis in the living cell.     

The dominating role of N-deacetylase/N-sulfotransferase 1 in forming domain structures in heparan sulfate

Heparan sulfate (HS) is a highly sulfated polysaccharide participated in essential physiological functions from regulating cell growth to blood coagulation. HS contains sulfated domains known as N-S domains and low sulfate domains known as N-Ac domains. The distribution of the domain structures is likely governed by the action of glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here, we sought to determine the substrate specificity of NDST using model substrates and recombinant NDST protein. We discovered that NDST-1 carries out the modification in a highly ordered fashion. The enzyme sulfates the substrate from the nonreducing end toward the reducing end consecutively, leading to the product with a cluster of N-sulfo glucosamine residues. Furthermore, a preexisting N-sulfo glucosamine residue prevents the action of NDST-1 at the residues immediately located at the nonreducing end, allowing the formation of an N-Ac domain. Our results provide the long sought evidence for understanding the formation of sulfated versus nonsulfated domains in the HS isolated from cells and tissues. The study demonstrates the regulating role of NDST-1 in mapping the sulfation patterns of HS.     

MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.     

Glucosaminyl N-deacetylase/N-sulphotransferases in heparan sulphate biosynthesis and biology

During the biosynthesis of heparan sulphate (HS) in the Golgi compartment, the first modification enzyme, glucosaminyl N-deacetylase/N-sulphotransferase (NDST), starts to work on the growing HS polysaccharide chain. This enzyme defines the overall design of the sulphation pattern, which will determine the ability of the HS chain to interact with target molecules. NDST removes acetyl groups from glucosamine residues and replaces them with sulphate groups. These N-sulphate groups are essential for further modification during biosynthesis; without N-sulphation, no O-sulphation or conversion of glucuronic acid into iduronic acid will occur. Four NDST isoforms, transcribed from four genes, have been identified. Much of our work is concentrated on how the enzymes are organized within the Golgi compartment and the identification of interacting partners. In addition, we study mice in which the gene encoding NDST-1 or NDST-2 has been knocked out. NDST-1 knockout mice with altered HS structure die at birth due to lung failure, whereas lack of NDST-2 results in abnormal mast cells. Since NDSTs have a key role in HS design (see above), these mice can be used to study HS function. Areas of interest are cell differentiation, growth, inflammation, cancer, lipid metabolism and microbial infection.     

Biosynthesis of heparan sulfate. Coordination of polymer-modification reactions in a Chinese hamster ovary cell mutant defective in N-sulfotransferase

A previous study identified a Chinese hamster ovary cell mutant, pgsE-606, which is defective in the N-sulfotransferase that catalyzes one of the initial polymer-modification reactions in the biosynthesis of heparan sulfate (Bame, K. J., and Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065). The structure of heparan sulfate generated by these cells reflects a 3-5-fold reduction in enzyme activity. The mutant produces heparan sulfate with half the content of N-sulfated glucosamine residues of that produced by wild-type cells and a more sparse distribution of N-sulfated residues. The present study demonstrates corresponding reductions in the proportion of 6-O-sulfated glucosamine residues (41% reduction) and the content of L-iduronic acid (51% reduction). The amount of 2-O-sulfated L-iduronic acid declines more dramatically (from 25% of total L-iduronic acid in the wild type to 8.4% in the mutant). Enzymatic assay of mixed O-sulfotransferases showed that the mutant has more activity than the wild type. Previous studies on the biosynthesis of heparin/heparan sulfate in cell-free systems point to a pivotal role of N-sulfation in determining the extent of the subsequent polymer-modification reactions. The present study shows that this concept also applies to heparan sulfate biosynthesis in the intact cell.     

Undersulfation of heparan sulfate restricts differentiation potential of mouse embryonic stem cells

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.     

Effect of substitution pattern on 1H, 13C NMR chemical shifts and 1J(CH) coupling constants in heparin derivatives

1H, 13C NMR chemical shifts and 1J(CH) coupling constants were measured for derivatives of heparin containing various sulfation patterns. 1H and 13C chemical shifts varied considerably after introducing electronegative sulfate groups. Chemical shifts of protons linked to carbons changed by up to 1 ppm on substitution with O- and N-sulfate or acetyl groups. Differences up to 10 ppm were detected for 13C chemical shifts in substituted glucosamine, but a less clear dependence was found in iduronate. 1J(CH) values formed two groups, corresponding to either sulfation or non-sulfation at positions 2 and 3 of glucosamine. O-sulfation caused increases up to 6 Hz in 1J(CH) and N-sulfation decreases up to 4 Hz. N-acetylation gave similar 1J(CH) values to N-sulfation. At positions 2 and 3 of iduronate the trend was less marked; 1J(CH) for O-sulfated positions usually increasing. Introduction of sulfate groups influences chemical shift and 1J(CH) values at the position of substitution, but also at more remote positions. 1J(CH) at the glycosidic linkage positions varied between free-amino and N-sulfated compounds, by up to 9 Hz. These results and changes in chemical shift values suggest that iduronate residues and the glycosidic linkages are affected, indicating overall conformational change. This may have important implications for biological activities.     

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.     

Glycosaminoglycan biosynthesis in arterial wall. Sulfation of heparan sulfate in cell membrane of aortic media-intima

Incubation of microsomal fractions with labelled 3'-phosphoadenylyl sulfate results in incorporation of [35S]sulfate into endogenous glycosaminoglycans. Specific radioactivity observed incorporated into heparan sulfate chains is 10-fold greater than that incorporated into chondro?tin sulfate chains. This is in agreement with the results obtained for glycosylation of glycosaminoglycans in arterial wall membrane fractions. Sulfation of heparan sulfate was studied since it contains N- and O-sulfate groups in contrast with the other sulfated glycosaminoglycans which contain only O-sulfate groups. Sulfation of heparan sulfate occurs rapidly, since sulfate incorporation is detected after exposure for only 0.5 min. Heparan sulfate was identified on the basis of its resistance to hyaluronidase and chondro?tin ABC lyase, its susceptibility to heparitinase, its sensitivity to nitrous acid and the presence of glucosamine as the only hexosamine. The chemical composition of the purified heparan sulfate fractions provides evidence for the high degree of sulfation of its chains. Studies into the distribution of sulfate residues on heparan sulfate at different times of sulfation indicate that N-sulfate groups are not randomly introduced into the polymer. The relationship between the processes of N- and O-sulfation was studied. The present results demonstrate that preferential N-sulfation is obtained for incorporation of labelled precursor over a short period, the O-sulfation occurring on previously N-sulfated heparan sulfate.     

Insights into the N-Sulfation Mechanism: Molecular Dynamics Simulations of the N-Sulfotransferase Domain of Ndst1 and Mutants

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS), yielding sulfated sugar chains and 3′-phosphoadenosine-5′-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.     

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.