Interaction Of Heparan Sulfate Research Articles

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

AbstractWe 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-1α 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-1α, 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-1α. 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-1α, 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)

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)

Mutations in the E2 Glycoprotein of Venezuelan Equine Encephalitis Virus Confer Heparan Sulfate Interaction, Low Morbidity, and Rapid Clearance from Blood of Mice

Mutations in the E2 Glycoprotein of Venezuelan Equine Encephalitis Virus Confer Heparan Sulfate Interaction, Low Morbidity, and Rapid Clearance from Blood of Mice

Cell surface heparan sulfate proteoglycans and lipoprotein metabolism.

Cell surface heparan sulfate proteoglycans are involved in several aspects of the lipoprotein metabolism. Most of the biological activities of these proteoglycans are mediated via interactions of their heparan sulfate moieties with various protein ligands, including lipoproteins and lipases. The binding of lipoproteins to heparan sulfate is largely determined by their apoprotein composition, and apoproteins B and E display the highest affinity for heparan sulfate. Interactions of lipoproteins with heparan sulfate are important for the cellular uptake and turnover of lipoproteins, in part by enhancing the accessibility of lipoproteins to lipoprotein receptors and lipases. Apoprotein B may interact with receptors without involving heparan sulfate. Heparan sulfate has been further implicated in presentation and stabilization of lipoprotein lipase and hepatic lipase on cell surfaces and in the transport of lipoprotein lipase from extravascular cells to the luminal surface of the endothelia. In atherosclerosis, heparan sulfate is intimately involved in several events important to the pathophysiology of the disease. Heparan sulfate thus binds and regulates the activity of growth factors, cytokines, superoxide dismutase and antithrombin, which contribute to aberrant cell proliferation, migration and matrix production, scavenging of reactive oxygen radicals and thrombosis. In this review we discuss the various roles of heparan sulfate proteoglycans in vascular biology, with emphasis on interactions of heparan sulfate with lipoproteins and lipases and the molecular basis of such interactions.

Inflammatory Mediators Regulate Cathepsin S in Macrophages and Microglia: A Role in Attenuating Heparan Sulfate Interactions

Cathepsin S is a member of the family of cysteine lysosomal proteases. The distribution of cathepsin S is restricted to cells from the mononuclear lineage both in the brain and in the periphery. Also, its protease activity is uniquely stable at neutral pH. We compared the expression of cathepsin S, B, and L mRNAs in various undifferentiated and differentiated cells of mononuclear origin, and examined the modulation of these mRNAs by inflammatory mediators (lipopolysaccharide and various cytokines). In addition, the effect of these agents on cathepsin S protein levels and protease activity was also determined. Lastly, the ability of cathepsin S to process basement membrane components such as heparan sulfate proteoglycans in vitro and in vivo was assessed. Cathepsin S, B, and L mRNAs are expressed in mature macrophages and microglial cells and not in undifferentiated monocytes. Activators of macrophages negatively regulate all three transcripts. Consistent with this, treatment with these agents leads to a decrease in intracellular cathepsin S protein levels and activity. However, the same treatments result in stimulation of secreted cathepsin S activity. Cathepsin S is capable of degrading heparan sulfate proteoglycans in vitro. Also, when expressed in endothelial cells, cathepsin S autocrinely attenuates the basic fibroblast growth factor (bFGF)-mediated binding of FGF receptor containing cells to endothelial cells, by acting on basement membrane proteoglycans. Taken together, these data imply that cathepsin S is a regulatable cysteine protease that plays a role in the degradation of extracellular proteins, whose secretion from macrophages and microglia is increased by signals that lead to activation of these cells, and may be important in regulating extracellular matrix interactions. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p320.html

Heparin Binding and Augmentation of C1 Inhibitor Activity

Heparin and other glycosaminoglycans have profound activityin vitroon the regulation of complement activity. The studies reported here examined the mechanism whereby heparin enhances C1 esterase inhibitor (C1INH) activity on C1 esterase (C1). The interaction of heparin and heparan sulfate with C1INH was first examined using surface plasmon resonance. Heparin was immobilized on a biosensor chip in two orientations, at its reducing end and in midchain, and heparan sulfate was immobilized at its reducing end. Heparin immobilized at its reducing end interacted with C1INH, giving an association constant (Ka) value of 1.43 × 107M−1, whereas heparin immobilized in midchain afforded aKavalue of 7 × 106M−1. No interaction between C1INH and heparan sulfate could be observed. Next, the augmentation of C1INH by heparin (Mr(av) 13,000), low-molecular-weight (LMW) heparin (Mr(av) 5000), and heparan sulfate (Mr(av) 11,000) was determined. C1INH alone was at least 10,000 times more active in inhibiting fluid phase C1 compared with erythrocyte-bound C1 (EAC1). When C1 was in the fluid phase, both heparin and LMW heparin were relatively ineffective at augmenting C1INH activity on C1. In contrast, when C1 was present as EAC1, heparin augmented C1INH activity at all C1INH concentrations examined and LMW heparin was up to 1.3 times more effective than heparin. This augmentation only occurred when both C1INH and heparin were present together with the EAC1. Hence, although surface plasmon resonance shows that heparin binds to C1INH, heparin augmentation of C1INH activity appears to require a terniary complex in which cell bound C1 interacts with both heparin and C1INH. This is the first report of LMW heparin augmenting C1INH activity. Heparan sulfate neither interacted with C1INH nor did it augment C1INH activity.

Heparan Sulfate Oligosaccharides Require 6-O-Sulfation for Promotion of Basic Fibroblast Growth Factor Mitogenic Activity

The interaction of heparan sulfate (HS) with basic fibroblast growth factor (bFGF) is influential in enabling the growth factor to bind to its cell surface tyrosine kinase receptor. In this study, we have investigated further the structural properties of HS required to mediate the activity of bFGF in a mitogenic assay. We have prepared a library of heparinase III-generated HS oligosaccharides fractionated by both their size (dp6-dp12) and sulfate content. The ability of these oligosaccharides to activate bFGF in a mitogenic assay was then correlated with their length and disaccharide composition. All octa- and hexasaccharide fractions tested were unable to activate bFGF. Dodeca- and decasaccharide fractions were found to contain both activating and non-activating oligosaccharides, and showed a clear correlation between total sulfate content and the level of activatory activity. Disaccharide analysis of a range of dodeca- and decasaccharide fractions showed that both activating and non-activating oligosaccharides were composed mainly of N-sulfated and IdoA(2S)-containing disaccharides. The only significant difference between activating and non-activating oligosaccharides was the content of 6-O-sulfated disaccharides, in particular the disaccharide IdoA(2S)alpha1,4GlcNSO3(6S). These results show that there is a requirement for 6-O-sulfation of N-sulfated glucosamine residues, in addition to the 2-O-sulfation of IdoA, for the promotion of bFGF mitogenic activity by naturally occurring HS oligosaccharides. Analysis of the structure-activity relationships in the dodecasaccharide fractions in particular, suggests that a minimum bFGF activation sequence exists which is dependent on the positioning of at least one 6-O-sulfate group.

Foam Cell Conversion of Macrophages Alters the Biosynthesis of Heparan Sulfate

Heparan sulfate is thought to regulate the biological activities of several proteins implicated in the pathogenesis of atherosclerosis. While the interactions of heparan sulfate with lipoprotein lipase and various growth factors have been actively studied, little is known of the cellular regulation of heparan sulfate biosynthesis in response to lipid accumulation. We have investigated heparan sulfate biosynthesis during conversion of murine J774 macrophages into lipid-laden foam cells. Such conversion is shown to accelerate the rate of glycosaminoglycan synthesis and the transport of newly synthesized proteoglycans into the medium. Moreover, the structure of heparan sulfate is specifically altered due to an ∼30% increase in the 6-O-sulfation of glucosamine residues within the N-sulfated heparan sulfate domains, whereas the sulfation of chondroitin sulfate remains unaffected. These results suggest a selective effect of foam cell conversion on the biosynthesis of heparan sulfate.

Interaction of Heparan Sulfate from Mammary Cells with Acidic Fibroblast Growth Factor (FGF) and Basic FGF: REGULATION OF THE ACTIVITY OF BASIC FGF BY HIGH AND LOW AFFINITY BINDING SITES IN HEPARAN SULFATE

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.

An Animal Cell Mutant Defective in Heparan Sulfate Hexuronic Acid 2-O-Sulfation

The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary cell mutant defective in 2-O-sulfation of iduronic acid. The mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic fibroblast growth factor (bFGF) to measure binding to cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.

Involvement of heparan sulfate and related molecules in sequestration and growth promoting activity of fibroblast growth factor.

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues [1, 2]. The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups [1, 2]. Beside serving as a scaffold for the attachment of various ECM components (e.g., collagen, laminin, fibronectin), the binding of HS to certain proteins has been suggested to induce a conformational change which may lead to the exposure of novel reactive determinants or conversely stabilize an inert protein configuration [1-4]. Of particular significance is the interaction of HS with fibroblast growth factors (FGFs), mediating their sequestration, stabilization and high affinity receptor binding and signaling [3-7]. Cellular responses to FGFs may hence be modulated by metabolic inhibitors of HS synthesis and sulfation, HS-degrading enzymes, and synthetic mimetics of heparin/HS. In the present review we focus on the involvement of HS in basic FGF (bFGF) receptor binding and mitogenic activity and its modulation by species of heparin, HS, and synthetic polyanionic 'heparin-mimicking' compounds. The results are discussed in relation to the current thoughts on the dual involvement of low and high affinity receptor sites in the growth promoting and angiogenic activities of bFGF and other heparin-binding growth factors.

Heparan sulfate staining of the glomerular basement membrane in relation to circulating anti-DNA and anti-heparan sulfate reactivity: a longitudinal study in NZB/W F1 mice.

Reactivity of serum antibodies with heparan sulfate (HS) has been associated with human and murine lupus nephritis, although the aetiological significance of this association is not clear. Recent work from our laboratories showed that binding of these antibodies to HS could be mediated by histone containing immune complexes. In human lupus nephritis we found a strong decrease in HS staining in the glomerular basement membrane (GBM). The aim of this study was to elucidate the correlation in experimental systemic lupus erythematosus (SLE) between albuminuria, staining of HS in the GBM and anti-DNA and anti-HS reactivity in plasma. We therefore studied NZB/W F1 mice during different stages of glomerular disease and compared them with age matched control NZB/W F1 mice without albuminuria. Anti-DNA and anti-HS reactivity were measured in longitudinally collected plasma samples and correlated with the onset of albuminuria, staining of HS in the glomerular basement membrane and deposition of immunoglobulins (Ig). HS staining was significantly decreased in the glomerular capillary loops of mice with prolonged proteinuria in comparison with age matched control mice (P = 0.0013). This decreased HS staining was correlated with increased Ig deposition in the capillary loops (tau = -0.42, P < 0.001), albuminuria (tau = -0.508, P < 0.001) and a decreased in anti-DNA levels measured in plasma (tau = 0.758, P < 0.005). Altered anti-HS reactivity in plasma did correlate with increased Ig deposition in the kidney (tau = 0.33, P < 0.05) but was not correlated with decreased staining of HS in the kidney. In conclusion, our study demonstrates that disappearance of staining of HS in the glomerular capillary loops is associated with albuminuria, increased Ig deposition in the glomerulus and decreased anti-DNA reactivity in plasma. Our findings are compatible with a model in which interaction ('masking') of HS with immune complexes consisting of anti-DNA antibodies and nucleosomes takes place.

Heparan sulfate initiates signals in murine macrophages leading to divergent biologic outcomes.

We have previously shown that the interaction of heparan sulfate (HS), a constituent of cell surfaces and extracellular matrices, with murine macrophages causes activation of the macrophages leading to the production of cytokines and PGE2 and profound changes in the cellular immune responses triggered by the macrophages. Here we describe the molecular mechanisms that underlie these immunoregulatory changes. We demonstrate that HS delivers signals to macrophages through at least two pathways, one involving the activation of a tyrosine kinase and of nuclear factor-KB, and the other involving the activation of protein kinase C and the elevation of intracellular calcium. The former pathway is associated with the production of IL-6, and the latter pathway is associated with the production of PGE2. Our findings suggest a model in which components of the microenvironment, such as HS, may determine the functional state of an APC, thereby modifying immune responses.

Immunolocalization of CD44 and heparan sulfate chains on the stratum intermedium and papillary layer in the rat enamel organ.

We studied the immunohistochemical localization of CD44 and heparan sulfate (HS) chains in rat enamel organ by confocal laser scanning microscopy and transmission electron microscopy. We also investigated the binding sites of basic fibroblast growth factor (bFGF), one of the heparin-binding growth factors (HBGF), on Microslicer-sections to clarify its role in the cell-cell interaction of HS. At the differentiation stage of ameloblast, weak immunoreactivity for CD44 was detected on the plasma membrane of the inner enamel epithelium, external enamel epithelium and the cells adjacent to the inner enamel epithelium. In accordance with the differentiation of preameloblasts into secretory ameloblasts, this immunoreactivity increased in stratum intermedium cells. At the secretory stage, stratum intermedium cells showed the most intense immunoreactivity in the enamel organ. At the maturation stage, strong immunoreactivity was seen on papillary layer cells. On the other hand, the lateral plasma membrane of ruffle-ended (RA) and smooth-ended ameloblast (SA) showed weak reactivity. No immunoreactivity was detected on the ruffled border of RA and the distal plasma membrane of SA. Immunolocalization of HS chains was similar to that of CD44. The binding activity of bFGF was also intense on stratum intermedium cells and papillary layer cells. These findings suggest that: 1) stratum intermedium cells and papillary layer cells express CD44 and HS chains in accordance with their differentiation; 2) HS chains on the plasma membrane of these cells may regulate calcium transport by their negative charge; and 3) HS chains on the stratum intermedium and papillary layer may play an important role in the differentiation and activity of ameloblasts by preserving HBGF.

Cells selected for high tumorigenicity or transformed by simian virus 40 synthesize heparan sulfate with reduced degree of sulfation.

Cell lines, selected from two independent clones of an established mouse embryo cell line by their ability to grow as solid tumors in immunocompetent syngeneic hosts, were found to have the same alteration in anion exchange properties as was previously reported for simian virus 40 (SV40)-transformed subclones. One tumor cell line (219CT) and one SV40-transformed subclone (215CSC) were selected for further detailed comparison with their common parent clone (210C). Cellulose acetate electrophoresis at pH 1.0 showed that 215CSC heparan sulfate had a slight overall decrease in sulfation compared with heparan sulfate from 210C; however, no gross difference in sulfation could be detected between heparan sulfate from 219CT and 210C. Analysis of the products of deaminative cleavage of heparan sulfate by nitrous acid under conditions where cleavage occurs quantitatively at N-sulfated glucosamine residues showed that, although heparan sulfate from the three cell lines gave similar yields of O-sulfated disaccharides, both 215CSC and 219CT had only about half as many O-sulfate residues in higher molecular weight oligosaccharides compared to heparan sulfate from 210C. Enzymatic degradation of heparan sulfate with a mixture of enzymes from Flavobacterium heparinum showed that this common alteration in heparan sulfate from both 215CSC and 219CT resulted from a 30% decrease in glucosamine residues bearing 6-O-sulfate groups. As this decrease in 6-O-sulfate glucosamine residues occurs in regions of the chain containing relatively few sulfate groups, it is clear that certain sequences of charged groups present in heparan sulfate frm 210C will be found only rarely in heparan sulfate from 215CSC and 219CT. It is suggested that this will result in alterations of the interaction of heparan sulfate with other molecules in the microenvironment at the cell surface which may be important in the control of such phenomena as cell growth and adhesion.