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At least 19 recordsLinked to original sources
PubMed2003

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.

Animals↗
PubMed2000

Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins.

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell "niches" in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAGs, 95 +/- 7%; P <.001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1alpha, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 +/- 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1alpha. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

Adult↗
PubMed2003

Mutations in the N termini of herpes simplex virus type 1 and 2 gDs alter functional interactions with the entry/fusion receptors HVEM, nectin-2, and 3-O-sulfated heparan sulfate but not with nectin-1.

Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-O-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-O-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether nectin-2 or 3-O-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors.

Amino Acid Sequence↗
PubMed2004

A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1.

Membrane fusion induced by herpes simplex virus (HSV) is required for both entry and cell-to-cell spread. It is mediated by the viral glycoprotein gB, gD, gH-gL and gD receptors. Although 3-O-sulfated heparan sulfate (3-OS HS) is a receptor for HSV-1 entry, the requirement for heparan sulfate in the fusion process has been ruled out. Here, it is demonstrated that cells expressing 3-OS HS, generated by D-glucosaminyl 3-O-sulfotransferase isoforms-3 and/or -5 (3-OST-3 and 3-OST-5), fused with cells expressing the four glycoproteins. The cell fusion observed exhibited similar requirements but was independent of protein receptors, HVEM or nectin-1. Additionally, removal of 3-OS HS from the cell surface by heparinase-I treatment and, in separate experiments, the presence of soluble 3-OST-3- and 3-OST-5-modified HS, significantly inhibited fusion. Taken together, these results indicate that 3-OS HS can play a crucial role in virus entry and cell fusion.

Animals↗
PubMed1984

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line.

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl-Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.

Animals↗
PubMed2000

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 </=50% of the iduronate residues. The degree of 2-O-sulfation was consistently increased with increasing NS domain length, suggesting a correlation between 2-O-sulfation efficiency and length of the polymeric substrate during heparan sulfate biosynthesis. By contrast, 6-O-sulfation of glucosamine units did not correlate to domain size. 6-O-Sulfation exceeded 2-O-sulfation in NS domains from kidney heparan sulfate, but was very low in aorta heparan sulfate. Remarkably, total O-sulfation of NS domains, i.e., the sum of 2-O- and 6-O-sulfate groups, was highly similar in all heparan sulfate samples investigated. The results reveal marked tissue-specific variation in the sulfation patterns of NS domains and indicate previously unrecognized distinctions in the coordination of the three polymer modification reactions during heparan sulfate biosynthesis.

Acetylglucosamine↗
PubMed1999

Isolation and characterization of a highly sulfated heparan sulfate from ascidian test cells.

Several sulfated polysaccharides have been isolated from the test cells of the ascidian Styela plicata. The preponderant polysaccharide is a highly sulfated heparan sulfate with the following disaccharide composition: (1) UA(2SO4)-1-->4 GlcN(SO4)(6SO4), 53%; (2) UA(2SO4)-1-->4-GlcN(SO4), 22%; (3) UA-1-->4-GlcNAc(6SO4), 14% and (4) UA-1-->4-GlcN(SO4), 11%. Two others unidentified sulfated polysaccharides and a glycogen polymer are also present in the ascidian eggs. Histochemistry with the cationic dye 1,9-dimethyl-methylene blue and biochemical analysis of the 35S-sulfate incorporation into the eggs reveal that the sulfated glycans are present exclusively in the test cells. Possibly these sulfated polysaccharides are involved in important functions of these cells, such as to confer an external and hydrophilic layer which protect the eggs and the larvae of ascidians.

Animals↗
PubMed1999

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.

Amino Acid Substitution↗
PubMed1997

Urinary chondroitin sulfates, heparan sulfate and total sulfated glycosaminoglycans in interstitial cystitis.

PURPOSE: We compared urinary glycosaminoglycan levels in patients with interstitial cystitis and healthy controls. MATERIALS AND METHODS: Total sulfated glycosaminoglycans assayed by dimethylmethylene blue binding and individual glycosaminoglycans analyzed by cellulose acetate electrophoresis were compared in patients with interstitial cystitis and healthy controls. Also, multiple urine samples were obtained from healthy female controls for 2 months to assess the relationship of urinary glycosaminoglycan and creatinine concentrations, and to determine whether glycosaminoglycan excretion changes during the menstrual cycle. RESULTS: Total sulfated glycosaminoglycan and creatinine concentrations correlated well in random voided samples. Menstrual cycle day did not affect total sulfated glycosaminoglycan levels. Cellulose acetate electrophoresis revealed 3 bands corresponding to chondroitin sulfates, heparan sulfate and acidic glycoprotein. Patients with interstitial cystitis had decreased urinary concentrations of each of these individual components and total sulfated glycosaminoglycans. However, glycosaminoglycan-to-creatinine ratios were similar in interstitial cystitis and control urine. CONCLUSIONS: Using these assays total and individual urinary glycosaminoglycan levels normalized to creatinine were not altered in interstitial cystitis.

Chondroitin Sulfates↗
PubMed1990

Comparative antithrombotic and hemorrhagic effects of dermatan sulfate, heparan sulfate and heparin.

Dermatan sulfate and heparan sulfate are currently under development as potential antithrombotic drugs. In our studies we have evaluated the relative antithrombotic and bleeding effects of these two agents in comparison to heparin, the commonly used anticoagulant. In a rabbit model of stasis thrombosis, a 500 micrograms/kg IV dose of dermatan or heparan produced 50-60% inhibition of induced in vivo thrombosis. At 750 micrograms/kg, both agents produced greater than 75% inhibition of thrombosis. Ex vivo measurement of plasma samples obtained from these animals demonstrated variable clotting effects at the lower dose and a proportional increase in the clotting activity at the higher dose. No anti-Xa or anti-IIa activity was observed in any sample. In contrast, animals treated with only 100 micrograms/kg heparin showed complete inhibition of induced thrombosis with significant anti-Xa and anti-IIa activities as well as prolongation of the clotting assays (APTT, TT and HeptestR). In the hemorrhagic studies utilizing a rabbit ear blood loss model, a 5.0 mg/kg IV dose of dermatan or heparan produced much less blood loss than heparin. On a gravimetric basis, dermatan and heparan were 10 fold less hemorrhagic than heparin. These results indicate that the relative contribution of plasmatic and cellular sites to the mediation of the antithrombotic action of heparin, dermatan and heparan differ. Although the antithrombotic dosages of dermatan and heparan are higher than heparin, due to the different mechanisms of action of each agent, a better safety index may be provided by dermatan and heparan than heparin.

Animals↗
PubMed2006

Role for 3-O-sulfated heparan sulfate as the receptor for herpes simplex virus type 1 entry into primary human corneal fibroblasts.

Herpes simplex virus type 1 (HSV-1) infection of the corneal stroma remains a major cause of blindness. Primary cultures of corneal fibroblasts (CF) were tested and found susceptible to HSV-1 entry, which was confirmed by deconvolution imaging of infected cells. Plaque assay and real-time PCR demonstrated viral replication and hence a productive infection of CF by HSV-1. A role for glycoprotein D (gD) receptors in cultured CF was determined by gD interference assay. Reverse transcription-PCR analysis indicated expression of herpesvirus entry mediator and 3-O-sulfated (3-OS) heparan sulfate (HS)-generating enzyme 3-O sulfotransferase 3 (3-OST-3) but not nectin-1 or nectin-2. Subsequently, HS isolated from these cells was found to contain two distinct disaccharides (IdoUA2S-AnMan3S and IdoUA2S-AnMan3S6S) that are representative of 3-OST-3 activity. The following lines of evidence supported the important role of 3-OS HS as the mediator of HSV-1 entry into CF. (i) Blockage of entry was observed in CF treated with heparinases. The same enzymes had significantly less effect on HeLa cells that use nectin-1 as the entry receptor. (ii) Enzymatic removal of cell surface HS also removed the major gD-binding receptor, as evident from the reduced binding of gD to cells. (iii) Spinoculation assay demonstrated that entry blockage by heparinase treatment included the membrane fusion step. (iv) HSV-1 glycoprotein-induced cell-to-cell fusion was inhibited by either prior treatment of cells with heparinases or by HS preparations enriched in 3-OS HS. Taken together, the data in this report provide novel information on the role of 3-OS HS in mediating infection of CF, a natural target cell type.

Animals↗
PubMed1996

Synchronized order of appearance of hyaluronic acid (or acidic galactan) --> chondroitin C-6 sulfate --> chondroitin C-4/C-6 sulfate, heparan sulfate, dermatan sulfate --> heparin during morphogenesis, differentiation and development.

The synthesis of glycosaminoglycans and acidic polysaccharides during embryonic and fetal development in mammals and molluscs is briefly reviewed. A sequential order of appearance of each of the acidic polysaccharides was observed, coinciding with the major processes of the ontogeny. In mammals, hyaluronic acid is the first glycosaminoglycan synthesized at the beginning of morphogenesis. This glycosaminoglycan is then replaced by chondroitin 6-sulfate during the migration of the mesenchymal cells. Heparan sulfate, dermatan sulfate and chondroitin 4-sulfate are synthesized only during cell differentiation. The synthesis of heparin, on the other hand, is confined to mast cells in a few tissues and is a late event in the differentiation process. The same general pattern is also observed in molluscs except that hyaluronic acid is replaced by an acidic galactan in the morphogenetic process. The activity of the degrading enzymes responsible for the disappearance of hyaluronic acid, chondroitin sulfate and the acidic galactan in each phase of embryonic development is also reviewed.

Animals↗
PubMed2006

Isolation and characterization of low sulfated heparan sulfate sequences with affinity for lipoprotein lipase.

Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains.

Acetylation↗
PubMed2006

N- and 6-O-sulfated heparan sulfates mediate internalization of coxsackievirus B3 variant PD into CHO-K1 cells.

Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO(3)(6OSO(3))](n), but not basic fibroblast growth factor binding to [HexUA-GlcNSO(3)-HexUA-GlcNSO(3)-IdUA(2OSO(3))](n), was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO(3)(6OSO(3))](n) is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.

Animals↗
PubMed2004

A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein: significance for malarial sporozoite attachment to hepatocytes.

Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP. We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity. Only one of these peptides (Pf 2), which contains a motif we have named region I-plus, demonstrated both high affinity heparin/HS binding activity and the ability to block the binding of recombinant CSP to heparin-Sepharose 4B. Analysis by isothermal titration calorimetry revealed that region I-plus has a binding constant of K(d) = 5.0 microm and a stoichiometry of n = 7.8 binding sites/heparin chain. Heparin binding was dependent on the amino acid sequence of region I-plus, and the binding sites on heparin/HS are contained within a decasaccharide. Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content (heparin-like) are required for efficient binding. Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I-plus/HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes. Finally, the region I-plus heparin/HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N-terminal portion of CSP.

Amino Acid Motifs↗