Regulation of heparan sulfate 6-O-sulfation by beta-secretase activity

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.     

Dromedary glycosaminoglycans: molecular characterization of camel lung and liver heparan sulfate

Glycosaminoglycans (GAGs) are the portion of a proteoglycan that determine its final shape and function. The molecular structure of predominant GAG species in camel liver and lung is reported for the first time. The one-humped camel survives in an extreme, arid habitat and, thus, offers a good model to study the role of glycomics on homeostasis. Heparan sulfate (HS) from the lung and liver of the one-humped camel were isolated. Characterization of these newly isolated glycosaminoglycans included (1)H NMR spectroscopy and disaccharide compositional analysis. The relative molecular weight of these GAGs was estimated by gradient polyacrylamide gel electrophoresis and their degree of sulfation was also assessed. Anticoagulant activity was determined using an anti-factor Xa assay and the HS from camel lung shows approximately 50% of heparin's activity. The structural differences of camel liver GAGs compared to human and porcine liver heparin and HS is discussed. Camel lung heparan sulfate resembles both heparin and HS in its structure and properties suggesting that it is either a highly sulfated form of HS, a mixture of heparin and HS or an undersulfated heparin.     

Host cell heparan sulfate glycosaminoglycans are ligands for OspF‐related proteins of the Lyme disease spirochete

Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous tissues. Host glycosaminoglycans, highly modified repeating disaccharides that are present on cell surfaces and in extracellular matrix, are common targets of microbial pathogens during tissue colonization. While several dermatan sulfate‐binding B. burgdorferi adhesins have been identified, B. burgdorferi adhesins documented to promote spirochetal binding to heparan sulfate have not yet been identified. OspEF‐related proteins (Erps), a large family of plasmid‐encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF‐related, OspEF‐leader peptide (Elp) and OspE‐related subfamilies. We show here that a member of the OspF‐related subfamily, ErpG, binds to heparan sulfate and when produced on the surface of an otherwise non‐adherent B. burgdorferi strain, ErpG promotes heparan sulfate‐mediated bacterial attachment to the glial but not the endothelial, synovial or respiratory epithelial cells. Six other OspF‐related proteins were capable of binding heparan sulfate, whereas representative OspE‐related and Elp proteins lacked this activity. These results indicate that OspF‐related proteins are heparan sulfate‐binding adhesins, at least one of which promotes bacterial attachment to glial cells.     

The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.     

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.     

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.     

The biosynthesis of anticoagulant heparan sulfate by the heparan sulfate 3-O-sulfotransferase isoform 5

The role of heparan sulfate (HS) in regulating blood coagulation has a wide range of clinical implications. In this study, we investigated the role of 3-O-sulfotransferase isoform 5 (3-OST-5) in generating anticoagulant HS in vivo. A Chinese hamster ovary cell line (3OST5/CHO) stably expressing 3-OST-5 was generated. The expression of 3-OST-5 in 3OST5/CHO cells was confirmed by Northern blot analysis, RT-PCR, and the disaccharide analyses of the HS from the cells. We also determined the effects of the HS from 3OST5/CHO on antithrombin-mediated inhibition of factor Xa. Fluorescently labeled antithrombin bound to the surface of 3OST5/CHO cells, suggesting that the antithrombin-binding HS is indeed present on the cell surface. Our results demonstrate that the 3-OST-5 gene is capable of synthesizing anticoagulant HS in CHO cells and has the potential to contribute to the biosynthesis of anticoagulant HS in humans.     

Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells

Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% +/- 8% for HS-deficient and 12% +/- 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.     

Regulation of basic fibroblast growth factor binding and activity by cell density and heparan sulfate

The role of cell density in modulating basic fibroblast growth factor binding and activity was investigated. A primary corneal stromal fibroblast cell culture system was used, since these cells do not constitutively express heparan sulfate proteoglycans in vivo except after injury. A 3-5-fold reduction in bFGF binding per cell was observed as cell density increased from 1000 to 35,000 cells/cm2. The cell density-dependent change in bFGF binding was not the result of altered FGFR expression as determined by equilibrium binding experiments and by immunoblot analysis. However, bFGF-cell surface receptor binding affinities were measured to be 10-20-fold higher at low cell densities than at intermediate and high cell density. bFGF-induced cell proliferation was also cell density-dependent, with maximal stimulation of proliferation 190-280% greater at intermediate densities (15,000 cells/cm2) than at other cell densities. This effect was specific to bFGF as serum, epidermal growth factor, and transforming growth factor-beta did not exhibit the same density-dependent profile. Further, heparan sulfate proteoglycans and, specifically, syndecan-4 were implicated as the modulator of bFGF binding and activity. Pretreatment of cell cultures with heparinase resulted in reduced bFGF binding to the cells and abrogated bFGF induced proliferation. These data suggest a mechanism by which cell density regulates heparan sulfate proteoglycan expression and modulates the cellular response to bFGF. Modulation of heparan sulfate proteoglycan expression might be an important aspect of the regulation of stromal cell migration and proliferation during wound healing.     

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.     

Biosynthesis of heparan sulfate

Microsomal preparations from Englebreth-Holm-Swarm mouse sarcoma were incubated with UDP-N-acetyl[³H] glucosamine and UDP-[¹⁴C]glucuronic acid to form proteoglycan containing [³H,¹⁴C]glycosaminoglycan with equimolar amounts of [³H]glucosamine and [¹⁴C]glucuronic acid. The labelled glycosaminoglycan was totally resistant to degradation by testicular hyaluronidase, but could be degraded readily by a crudeFlavobacter heparinum enzyme preparation which is capable of degrading heparin and heparan sulfate. Chromatography of the [³H,¹⁴C]glycosaminoglycan on DEAE-cellulose provided a pattern with three peaks: the first appearing before hyaluronic acid, the second and largest appearing at the site of hyaluronic acid, and a third appearing slightly beyond hyaluronic acid but before a standard of chondroitin sulfate. When 3-phosphoadenosine 5-phosphosulfate was also included in the reaction mixture, a change appeared in the [³H,¹⁴C]glycosaminoglycan so that chromatography on DEAE-cellulose presented a pattern with a significant amount of material which cochromatographed in the area where heparan sulfate would be found. There was no material that co-chromatographed with the more highly sulfated substance, heparin. This indicates that the microsomal preparation from the Englebreth-Holm-Swarm sarcoma is capable of producing a heparan sulfate-like molecule and is controlled in its sulfation of precursors so that heparin is not formed.     

Increased permeability of the glomerular basement membrane to ferritin after removal of glycosaminoglycans (heparan sulfate) by enzyme digestion

Glomerular basement membranes (GBM's) were subjected to digestion in situ with glycosaminoglycan-degrading enzymes to assess the effect of removing glycosaminoglycans (GAG) on the permeability of the GBM to native ferritin (NF). Kidneys were digested by perfusion with enzyme solutions followed by perfusion with NF. In controls treated with buffer alone, NF was seen in high concentration in the capillary lumina, but the tracer did not penetrate to any extent beyond the lamina rara interna (LRI) of the GBM, and litte or no NF reached the urinary spaces. Findings in kidneys perfused with Streptomyces hyaluronidase (removes hyaluronic acid) and chondroitinase-ABC (removes hyaluronic acid, chondroitin 4- and 6-sulfates, and dermatan sulfate, but not heparan sulfate) were the same as in controls. In kidneys digested with heparinase (which removes most GAG including heparan sulfate), NF penetrated the GBM in large amounts and reached the urinary spaces. Increased numbers of tracer molecules were found in the lamina densa (LD) and lamina rara externa (LRE) of the GBM. In control kidneys perfused with cationized ferritin (CF), CF bound to heparan- sulfate rich sites demonstrated previously in the laminae rarae; however, no CF binding was seen in heparinase-digested GBM's, confirming that the sites had been removed by the enzyme treatment. The results demonstrated that removal of heparan sulfate (but not other GAG) leads to a dramatic increase in the permeability of the GBM to NF.     

A conformational change in heparan sulfate 3-O-sulfotransferase-1 is induced by binding to heparan sulfate

The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.     

Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.     

Regulation of cell volume by glycosaminoglycans

Cell volume is regulated by a delicate balance between ion distribution across the plasma membrane and the osmotic properties of intra‐ and extracellular components. Using a fluorescent calcein indicator, we analysed the effects of glycosaminoglycans on the cell volume of hyaluronan producing fibroblasts and hyaluronan deficient HEK cells over a time period of 30 h. Exogenous glycosaminoglycans induced cell blebbing after 2 min and swelling of fibroblasts to about 110% of untreated cell volume at low concentrations which decreased at higher concentrations. HEK cells did not show cell blebbing and responded by shrinking to 65% of untreated cell volume. Heparin induced swelling of both fibroblasts and HEK cells. Hyaluronidase treatment or inhibition of hyaluronan export led to cell shrinkage indicating that the hyaluronan coat maintained fibroblasts in a swollen state. These observations were explained by the combined action of the Donnan effect and molecular crowding. J. Cell. Biochem. 113: 340–348, 2012. © 2011 Wiley Periodicals, Inc.     

The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.     

Dietary manganese affects the concentration, composition and sulfation pattern of heparan sulfate glycosaminoglycans in Sprague-Dawley rat aorta

We examined the effect of dietary Mn on the composition and structure of heparan sulfate (HS) glycosaminoglycans (GAGs) of rat aorta. Animals were randomly assigned to either a Mn deficient (MnD), adequate (MnA) or supplemented (MnS) diet (Mn<1, 10–15 and 45–50 ppm, respectively). After 15 weeks, aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition of the HS fractions was determined by HPCE following treatment with heparin lyases I, II and III. In MnS aortas we observed increased concentration of total GalAGs and decreased concentration of HS and HA, when compared to MnA aortas. Aortas from MnD and MnA rats appeared to have similar distribution of individual GAGs. Heparan sulfate chains of MnS aortas contained higher (41%) concentration of non-sulfated units compared to MnA ones. Variable amounts of trisulfated and disulfated units were found only in MnD and MnA groups but not in MnS. Our results demonstrate that HS biosynthesis in the rat aorta undergoes marked structural modifications that depend upon dietary Mn intake. The reduced expression and undersulfation of HSPGs with Mn supplementation might indicate a reduced ability of vascular cells to interact with biologically active molecules such as growth factors. Alterations in cell-membrane binding ability to a variety of extracellular ligands might affect signal-transduction pathways and arterial functional properties.