A single protein catalyzes both N-deacetylation and N-sulfation during the biosynthesis of heparan sulfate

Heparan sulfate is a highly sulfated carbohydrate polymer that binds to and modulates the activities of numerous proteins. The formation of these protein-binding domains in heparan sulfate is dependent on a series of biosynthetic reactions that modify the polysaccharide backbone; the initiating and rate-limiting steps of this process are the N-deacetylation and N-sulfation of N-acetylglucosamine residues in the polymer. We now report that in the rat liver, biosynthesis of heparan sulfate utilizes a single protein that possesses both N-deacetylase and N-sulfotransferase activities. This was accomplished by demonstrating that both activities resided in a purified soluble fusion protein containing the Golgi-lumenal portion of the enzyme. We propose that this protein be renamed the rat liver Golgi heparan sulfate N-deacetylase/N-sulfotransferase.     

Binding of Sindbis virus to cell surface heparan sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.     

Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of the major heparan sulfate structural determinants

We have demonstrated by affinity chromatography that hepatocyte growth factor (HGF) binds strongly to heparan sulfate (HS). This substantiates previous suggestions that cell-surface heparan sulfate proteoglycans constitute the so-called low affinity cellular binding sites for HGF. Using a recombinant human HGF affinity column, we have analyzed the effects of various specific chemical and enzymatic modifications/depolymerizations of HS on its affinity in order to elucidate the polysaccharide structural determinants. Interaction is shown to be only slightly affected by digestion with heparinase I or III or by replacement of N-sulfates with N-acetyl groups. This suggests a specific role for sulfated domains containing nonsulfated IdceA residues, with only a small contribution from N-sulfates and IdceA(2-OSO3) residues. In addition, disaccharide analyses of various HGF-binding oligosaccharides indicate that affinity is more closely associated with 6-O-sulfation of GlcNSO3 residues than with sulfation at any other position. Although interaction can be demonstrated with heparinase III-resistant oligosaccharides as small as hexasaccharides, the highest affinity was found with oligosaccharides containing a minimum of 10-12 monosaccharides. The structural specificity of the HGF-HS interaction is thus shown to be radically different from that previously described for the basic fibroblast growth factor-HS interaction.     

Extracellular matrix heparan sulfate modulates endothelial cell susceptibility to Staphylococcus aureus

We have examined several types of tumor cell lines and shown that they invariably expressed little or no Egr-1, in contrast to their normal counterparts. We have previously shown that the expression of exogenous Egr-1 in human breast and other tumor cells markedly reduces transformed growth and tumorigenicity. We therefore hypothesized that the loss of Egr-1 expression plays a role in transformation. All human and mouse breast cancer cell lines and tumors examined had reduced Egr-1 expression compared with their normal counterparts. Reduced Egr-1 expression was also observed in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, and this level increased to normal levels in tumors that regressed after tamoxifen treatment. We concluded, therefore, that loss of Egr-1 expression may play a role in the deregulation of normal growth in the tumorigenic process and that Egr-1 acts as a tumor suppressor gene.     

Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate

In this report, we demonstrate that the initial event in human cytomegalovirus (HCMV) infection is attachment to extracellular heparan sulfate. Further, this interaction is important for initiation of infection in fibroblast cells. Using microbinding assays to specifically monitor virus attachment as well as plaque titration assays to measure infectivity, we found that heparin competition as well as enzymatic digestion of cells with heparinase blocked virus attachment, initiation of immediate-early gene expression and infectivity. Other major glycosaminoglycans were found not to be involved in HCMV attachment and infectivity. In addition, HCMV was unable to attach to mutant derivatives of Chinese hamster ovary cells deficient in synthesis of heparan sulfate proteoglycans. Basic fibroblast growth factor, which requires initial interaction with extracellular heparin prior to binding to its high affinity receptor, also inhibited HCMV attachment to cells. Time-course experiments revealed that the initial HCMV binding was sensitive to heparin competition (10 micrograms/ml) or 0.75 M salt washes. The initial heparin-dissociable binding converted rapidly to high affinity (heparin resistant) HCMV attachment. These data suggest that sequential receptor interactions may mediate HCMV adsorption to cells. Heparin affinity chromatography revealed that multiple HCMV envelope glycoproteins, including gB, are capable of binding to heparin.     

Defining the interleukin-8-binding domain of heparan sulfate

Interleukin-8, a member of the CXC chemokine family, has been shown to bind to glycosaminoglycans. It has been suggested that heparan sulfate on cell surfaces could provide specific ligand sites on endothelial cells to retain the highly diffusible inflammatory chemokine for presentation to leukocytes. By using selectively modified heparin and heparan sulfate fragments in a nitrocellulose filter trapping system, we have analyzed sequence requirements for interleukin-8 binding to heparin/heparan sulfate. We demonstrate that the affinity of a monomeric interleukin-8 molecule for heparin/heparan sulfate is too weak to allow binding at physiological ionic strength, whereas the dimeric form of the protein mediates binding to two sulfated domains of heparan sulfate. These domains, each an N-sulfated block of approximately 6 monosaccharide units, are contained within an approximately 22-24-mer sequence and are separated by a region of 相似文献   

Characterization of the heparan sulfate and chondroitin sulfate assembly sites in CD44

Isoforms of CD44 are differentially modified by the glycosaminoglycans (GAGs) chondroitin sulfate (CS), heparan sulfate (HS), and keratan sulfate. GAG assembly occurs at serines followed by glycines (SG), but not all SG are utilized. Seven SG motifs are distributed in five CD44 exons, and in this paper we identify the HS and CS assembly sites that are utilized in CD44. Not all the CD44 SG sites are modified. The SGSG motif in CD44 exon V3 is the only HS assembly site; this site is also modified with CS. HS and CS attachment at that site was eliminated by mutation of the serines in the V3 motif to alanine (AGAG). Exon E5 is the only other CD44 exon that supports GAG assembly and is modified with CS. Using a number of recombinant CD44 protein fragments we show herein that the eight amino acids located downstream of the SGSG site in V3 are responsible for the specific addition of HS to this site. If the eight amino acids located downstream from the first SG site in CD44 exon E5 are exchanged with those located downstream of the SGSG site in exon V3, the SG site in E5 becomes modified with HS and CS. Likewise if the eight amino acids found downstream from the first SG in E5 are placed downstream from the SGSG in V3, this site is modified with CS but not HS. We also show that these sequences cannot direct the modification of CD44 with HS from a distance. Constructs containing CD44 exon V3 in which the SGSG motif was mutated to AGAG were not modified with HS even though they contained other SG motifs. Thus, a number of sequence and structural requirements that dictate GAG synthesis on CD44 have been identified.     

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.     

Age-dependent modulation of heparan sulfate structure and function

Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated L-iduronic acid (2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.     

Functional analysis of conserved cysteines in heparan sulfate N-deacetylase-N-sulfotransferases

N-Deacetylase-N-sulfotransferases (NDANST) catalyze the two initial modifications of the polysaccharide precursor in the biosynthesis of heparin and heparan sulfate. These modifications are the gating steps in establishing growth factor protein-binding domains of these glycosaminoglycans. We have undertaken a structure-activity analysis of the 841-amino acid Golgi-luminal portion of the rat liver NDANST to localize the two enzymatic functions. Each activity can be assayed in vitro independently of the other when provided with the appropriate substrate, and N-ethylmaleimide treatment selectively inactivates the deacetylase activity. In this study, dithiothreitol treatment of the rat liver NDANST was shown to inactivate the sulfotransferase function, while stimulating deacetylase activity 2-3-fold over the native protein. Site-directed mutagenesis of the eight cysteine (Cys) residues in the rat liver NDANST that are conserved in the mouse mastocytoma protein produced three important findings regarding the localization of each enzymatic function: 1) derivatization of Cys486 with N-ethylmaleimide resulted in total inactivation of the deacetylase activity based on steric hindrance of the active site (this residue was shown not to be involved in enzymatic catalysis), 2) substitution of either Cys159 or Cys486 with alanine resulted in enhanced activity of the deacetylase to the level obtained by dithiothreitol treatment, and 3) alanine substitution of Cys818 or Cys828 completely inactivated the sulfotransferase activity, while substitution of Cys586 or Cys601 resulted in a 90% loss in activity. These findings suggest that the two enzymatic domains within the NDANST localize to different portions of the protein, with two disulfide pairs toward the COOH-terminal half of the protein necessary for the sulfotransferase activity, and Cys residues within the NH2-terminal half influencing or located near the active site of the deacetylase functionality.     

Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface heparan sulfate proteoglycans

To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Chemoenzymatic synthesis of classical and non-classical anticoagulant heparan sulfate polysaccharides

Heparan sulfate (HS) polysaccharides interact with numerous proteins at the cell surface and orchestrate many different biological functions. Though many functions of HS are well established, only a few specific structures can be attributed to HS functions. The extreme diversity of HS makes chemical synthesis of specific bioactive HS structures a cumbersome and tedious undertaking that requires laborious and careful functional group manipulations. Now that many of the enzymes involved in HS biosynthesis are characterized, we show in this study how one can rapidly and easily assemble bioactive HS structures with a set of cloned enzymes. We have demonstrated the feasibility of this new approach to rapidly assemble antithrombin III-binding classical and non-classical anticoagulant polysaccharide structures for the first time.     

Regulation of anti-HIV-1 activity of RANTES by heparan sulfate proteoglycans

The role of cell surface proteoglycans in CC chemokine-mediated anti-HIV-1 activity in T cells and macrophages was investigated. Enzyme digestion of heparan sulfate (HS), but not chondroitin sulfate, from the surface of PM1(CD26H) cells (a human T cell line selected for high CD26 expression) rendered them resistant to the antiviral effects of RANTES and macrophage-inflammatory protein-1beta at otherwise inhibitory chemokine concentrations. HIV-1 infection of macrophages, however, was inhibited only partially, even at high concentrations of RANTES, and this inhibition was not prevented by HS removal. Flow cytometry revealed that digestion of cell surface proteoglycans, including HS, prevented the binding of RANTES at 10 to 100 nM concentrations to PM1(CD26H) cells. However, the binding of RANTES to activated macrophages occurred only at higher concentrations (100-300 nM) and was mostly chondroitin sulfate, and not HS, dependent. These results support a role for HS in facilitating the interaction of CC chemokines with the cell surface and the consequent inhibition of HIV-1 infection. The absence of HS-dependent binding of RANTES at lower concentrations to macrophages is consistent with the resistance of these cells to the antiviral effects of chemokines.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

Interaction of angiogenic basic fibroblast growth factor with endothelial cell heparan sulfate proteoglycans. Biological implications in neovascularization

Even if there were antiretrovirals developed that could completely eliminate HIV from the body, it is thought that immune-based therapy would still be necessary. Pervasive damage occurs in the immune system even in early stages of the disease, and this damage would not be corrected by antiretrovirals. Several different types of immune-based therapies are presented in this article; some have been successful, and some have not been successful. All have been important, however, in increasing the knowledge base of HIV pathogenesis and in narrowing the options that might rebuild the immune system and, thereby, reverse this pathology.     

The interaction of the transforming growth factor-betas with heparin/heparan sulfate is isoform-specific

We have undertaken a comparative study of the interaction of the three mammalian transforming growth factor-betas (TGF-beta) with heparin and heparan sulfate. TGF-beta1 and -beta2, but not -beta3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-beta1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-beta1 by alpha2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-beta2.alpha2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-beta3 cannot be affected. Comparison of the amino acid sequences of the TGF-beta isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-beta dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain.     

Syndecans, heparan sulfate proteoglycans, maintain the proteolytic balance of acute wound fluids

An imbalance between proteases and antiproteases is thought to play a role in the inflammatory injury that regulates wound healing. The activities of some proteases and antiproteases found in inflammatory fluids can be modified in vitro by heparin, a mast cell-derived glycosaminoglycan. Because syndecans, a family of cell surface heparan sulfate proteoglycans, are the major cellular source of heparin-like glycosaminoglycan, we asked whether syndecans modify protease activities in vivo. Syndecan-1 and syndecan-4 ectodomains are shed into acute human dermal wound fluids (Subramanian, S. V., Fitzgerald, M. L., and Bernfield, M. (1997) J. Biol. Chem. 272, 14713-14720). Moreover, purified syndecan-1 ectodomain binds cathepsin G (Kd = 56 nM) and elastase (Kd = 35 nM) tightly and reduces the affinity of these proteases for their physiological inhibitors. Purified syndecan-1 ectodomain protects cathepsin G from inhibition by alpha1-antichymotrypsin and squamous cell carcinoma antigen 2 and elastase from inhibition by alpha1-proteinase inhibitor by decreasing second order rate constants for protease-antiprotease associations (kass) by 3700-, 32-, and 60-fold, respectively. Both enzymatic degradation of heparan sulfate and immunodepletion of the syndecan-1 and -4 in wound fluid reduce these proteolytic activities in the fluid, indicating that the proteases in the wound environment are regulated by interactions with syndecan ectodomains. Thus, syndecans are shed into acute wound fluids, where they can modify the proteolytic balance of the fluid. This suggests a novel physiological role for these soluble heparan sulfate proteoglycans.