Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.     

Oligosaccharide Substrate Preferences of Human Extracellular Sulfatase Sulf2 Using Liquid Chromatography-Mass Spectrometry Based Glycomics Approaches

Sulfs are extracellular endosulfatases that selectively remove the 6-O-sulfate groups from cell surface heparan sulfate (HS) chain. By altering the sulfation at these particular sites, Sulfs function to remodel HS chains. As a result of the remodeling activity, HSulf2 regulates a multitude of cell-signaling events that depend on interactions between proteins and HS. Previous efforts to characterize the substrate specificity of human Sulfs (HSulfs) focused on the analysis of HS disaccharides and synthetic repeating units. In this study, we characterized the substrate preferences of human HSulf2 using HS oligosaccharides with various lengths and sulfation degrees from several naturally occurring HS sources by applying liquid chromatography mass spectrometry based glycomics methods. The results showed that HSulf2 preferentially digests highly sulfated HS oligosaccharides with zero acetyl groups and this preference is length dependent. In terms of length of oligosaccharides, HSulf2 digestion induced more sulfation decrease on DP6 (DP: degree of polymerization) compared to DP2, DP4 and DP8. In addition, the HSulf2 preferentially digests the oligosaccharide domain located at the non-reducing end (NRE) of the HS and heparin chain. In addition, the HSulf2 digestion products were altered only for specific isomers. HSulf2 treated NRE oligosaccharides also showed greater decrease in cell proliferation than those from internal domains of the HS chain. After further chromatographic separation, we identified the three most preferred unsaturated hexasaccharide for HSulf2.     

Heparan Sulfate Domain Organization and Sulfation Modulate FGF-induced Cell Signaling

Heparan sulfates (HSs) modulate various developmental and homeostatic processes by binding to protein ligands. We have evaluated the structural characteristics of porcine HS in cellular signaling induced by basic fibroblast growth factor (FGF2), using CHO745 cells devoid of endogenous glycosaminoglycans as target. Markedly enhanced stimulation of cell signaling, measured as phosphorylation of ERK1/2 and protein kinase B, was only observed with the shortest HS chains isolated from liver, whereas the longer chains from either liver or intestine essentially prolonged duration of signals induced by FGF2 in the absence of polysaccharide. Structural analysis showed that contiguous sulfated domains were most abundant in the shortest HS chains and were more heavily sulfated in HS from liver than in HS from intestine. Moreover, the shortest chains from either source entered into ternary complexes with FGF2 and FGF receptor-1c more efficiently than the corresponding longer chains. In addition to authentic HSs, decasaccharide libraries generated by chemo-enzymatic modification of heparin were probed for effect on FGF2 signaling. Only the most highly sulfated decamers, previously found most efficient in ternary complex formation (Jastrebova, N., Vanwildemeersch, M., Rapraeger, A. C., Giménez-Gallego, G., Lindahl, U., and Spillmann, D. (2006) J. Biol. Chem. 281, 26884–26892), promoted FGF2 cellular signaling as efficiently as short HS chains from liver. Together these results suggest that the effects of HS on FGF2 signaling are determined by both the structure of the highly sulfated domains and by the organization/availability of such domains within the HS chain. These findings underpin the need for regulation of HS biosynthesis in relation to control of growth factor-induced signaling pathways.     

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4⁺ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.     

Role of 6-O-Sulfated Heparan Sulfate in Chronic Renal Fibrosis

Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-β induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.     

Endothelial Heparan Sulfate 6-O-Sulfation Levels Regulate Angiogenic Responses of Endothelial Cells to Fibroblast Growth Factor 2 and Vascular Endothelial Growth Factor

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF₁₆₅) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF₁₆₅/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1–40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF₁₆₅-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF₁₆₅-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents.     

The low sulfated chondroitin sulfate proteoglycans of human placenta have sulfate group-clustered domains that can efficiently bind Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with approximately 30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only approximately 8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2-3% and 9-14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20-28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation.     

Glycomics analysis of mammalian heparan sulfates modified by the human extracellular sulfatase HSulf2

Background

The Sulfs are a family of endosulfatases that selectively modify the 6O-sulfation state of cell-surface heparan sulfate (HS) molecules. Sulfs serve as modulators of cell-signaling events because the changes they induce alter the cell surface co-receptor functions of HS chains. A variety of studies have been aimed at understanding how Sulfs modify HS structure, and many of these studies utilize Sulf knockout cell lines as the source for the HS used in the experiments. However, genetic manipulation of Sulfs has been shown to alter the expression levels of HS biosynthetic enzymes, and in these cases an assessment of the fine structural changes induced solely by Sulf enzymatic activity is not possible. Therefore, the present work aims to extend the understanding of substrate specificities of HSulf2 using in vitro experiments to compare HSulf2 activities on HS from different organ tissues.

Methodology/Principal Findings

To further the understanding of Sulf enzymatic activity, we conducted in vitro experiments where a variety of mammalian HS substrates were modified by recombinant human Sulf2 (HSulf2). Subsequent to treatment with HSulf2, the HS samples were exhaustively depolymerized and analyzed using size-exclusion liquid chromatography-mass spectrometry (SEC-LC/MS). We found that HSulf2 activity was highly dependent on the structural features of the HS substrate. Additionally, we characterized, for the first time, the activity of HSulf2 on the non-reducing end (NRE) of HS chains. The results indicate that the action pattern of HSulf2 at the NRE is different compared to internally within the HS chain.

Conclusions/Significance

The results of the present study indicate that the activity of Sulfs is dependent on the unique structural features of the HS populations that they edit. The activity of HSulf2 at HS NREs implicates the Sulfs as key regulators of this region of the chains, and concomitantly, the protein-binding events that occur there.     

Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.     

Purification and characterization of heparan sulfate from human primary osteoblasts

Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts—soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non‐identical by a number of parameters, and that these differences have significant ramifications for their ligand‐binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS‐dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132–1142, 2009. © 2009 Wiley‐Liss, Inc.     

Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin

We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units.     

Identification and characterization of a sulfoglycosidase from Bifidobacterium bifidum implicated in mucin glycan utilization

Human gut symbiont bifidobacteria possess carbohydrate-degrading enzymes that act on the O-linked glycans of intestinal mucins to utilize those carbohydrates as carbon sources. However, our knowledge about mucin type O-glycan degradation by bifidobacteria remains fragmentary, especially regarding how they decompose sulfated glycans, which are abundantly found in mucin sugar-chains. Here, we examined the abilities of several Bifidobacterium strains to degrade a sulfated glycan substrate and identified a 6-sulfo-β-d-N-acetylglucosaminidase, also termed sulfoglycosidase, encoded by bbhII from Bifidobacterium bifidum JCM 7004. A recombinant BbhII protein showed a substrate preference toward 6-sulfated and 3,4-disulfated N-acetylglucosamines over non-sulfated and 3-sulfated N-acetylglucosamines. The purified BbhII directly released 6-sulfated N-acetylglucosamine from porcine gastric mucin and the expression of bbhII was moderately induced in the presence of mucin. This de-capping activity may promote utilization of sulfated glycans of mucin by other bacteria including bifidobacteria, thereby establishing the symbiotic relationship between human and gut microbes.     

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.     

Apical Membrane Expression of Distinct Sulfated Glycans Is a Characteristic Feature of Ductules and Their Reactive and Neoplastic Counterparts

Intrahepatic bile ducts transport bile between bile canaliculi and the extrahepatic bile duct. The luminal surface of this tract is lined by a layer of biliary epithelial cells, or cholangiocytes, which secrete mucins consisting of scaffold proteins and O-glycosidically linked carbohydrate side chains. Although mucin core proteins have been extensively investigated, the structure and function of carbohydrate side chains have not. Here, we demonstrate that distinct sulfated glycans positive for MECA-79, R-10G, and 297-11A, but not 5D4, monoclonal antibodies are expressed in the cytoplasm of cells of large-sized ducts and in the apical membrane of cells in ductules, and that R-10G immunolabeling is partially eliminated by endo-β-galactosidase digestion, supporting the presence of N-acetylglucosamine-6-O-sulfated N-acetyllactosamine structures. We observed comparable apical membrane-predominant staining in ductular reactions seen during regeneration that occurs in various liver diseases and in cholangiolocarcinoma, a subtype of small duct-type intrahepatic cholangiocarcinoma (iCCA). Apical membrane expression of distinct sulfated glycans in large duct-type iCCA was negligible. Intriguingly, under pathological conditions, endo-β-galactosidase digestion almost completely eliminated R-10G immunoreactivity. These findings suggest that apical membrane expression of distinct sulfated glycans is a characteristic feature of ductules and their reactive and neoplastic counterparts     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.     

Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable

Many of the biological functions attributed to cell surface heparan sulfate (HS) proteoglycans, including the Syndecan family, are elicited through the interaction of their HS chains with soluble extracellular molecules. Tightly controlled, cell-specific sulfation and epimerization of HS precursors endows these chains with highly sulfated, iduronate-rich regions, which are major determinants of cytokine and matrix-protein binding and which are interspersed by N-acetylated, poorly sulfated regions. Until this study, there have been no comprehensive structural comparisons made on HS chains decorating simultaneously expressed, but different, syndecan core proteins. In this paper we demonstrate that the HS chains on affinity-purified syndecan-1 and -4 from murine mammary gland cells are essentially identical by a number of parameters. Size determination, disaccharide analyses, enzymatic and chemical scission methods, and affinity co-electrophoresis all failed to reveal any significant differences in fine structure, domain organization, or ligand-binding properties of these HS species. These findings lead us to suggest that the imposition of the fine structure onto HS occurs independently of the core protein to which it is attached and that these core proteins, in addition to the HS chains, may play a pivotal role in the various biological functions ascribed to these macromolecules.     

Heparan Sulfate Inhibits Hematopoietic Stem and Progenitor Cell Migration and Engraftment in Mucopolysaccharidosis I

Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for α-l-iduronidase. Idua^−/− mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua^−/− recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua^−/− BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua^−/− BM and specifically 2-O-sulfated HS, elevated in Idua^−/− BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease.     

The Synergy of 6-O-Sulfation and N- or 3-O-Sulfation of Chitosan Is Required for Efficient Inhibition of P-Selectin-Mediated Human Melanoma A375 Cell Adhesion

We prepared chitosan sulfated derivatives to address the common structural requirement of the sulfate pattern to block P-selectin-mediated tumor cell adhesion. Our results indicate that 6-O-sulfation of chitosan is indispensable for inhibition of P-selectin binding to human melanoma A375 cells. Furthermore, additional N-sulfation or 3-O-sulfation dramatically enhanced the inhibitory activity of 6-O-sulfated chitosan, suggesting that efficient anti-P-selectin adhesion activity of sulfated saccharides requires the synergy of 6-O-sulation and N- or 3-O-sulfation in glucosamine units.     

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.     

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

Sodium butyrate, a histone deacetylase inhibitor, has been used to improve transgene expression in Chinese hamster ovary (CHO) cells. The current study explores the impact of butyrate treatment on heparan sulfate (HS) biosynthesis and structural composition in a recombinant CHO-S cell line expressing enzymes in the heparin (HP)/(HS) biosynthetic pathway (Dual-10 stably expressing NDST2 and HS3st1). Flow cytometric analysis showed that antithrombin binding was increased in Dual-10 cells and basic fibroblast growth factor binding was decreased in response to sodium butyrate treatment. The results were in agreement with the AMAC-LCMS (2-aminoacridine-tagged HS/HP analysis by liquid chromatography mass spectrometry) data that showed that there was an increase in heparan sulfate tri-sulfated disaccharides and a decrease in N-sulfated disaccharides in the butyrate-treated cells. However, we could not detect any changes in the chondroitin sulfate pathway in Dual-10 cells treated with butyrate. The current study is the first to report the effect of butyrate on glycosaminoglycan profiles.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-013-9677-9) contains supplementary material, which is available to authorized users.