N-Sulfation of Heparan Sulfate Regulates Early Branching Events in the Developing Mammary Gland

Branching morphogenesis, a fundamental process in the development of epithelial organs (e.g. breast, kidney, lung, salivary gland, prostate, pancreas), is in part dependent on sulfation of heparan sulfate proteoglycans. Proper sulfation is mediated by biosynthetic enzymes, including exostosin-2 (Ext2), N-deacetylase/N-sulfotransferases and heparan sulfate O-sulfotransferases. Recent conditional knockouts indicate that whereas primary branching is dependent on heparan sulfate, other stages are dependent upon selective addition of N-sulfate and/or 2-O sulfation (Crawford, B .E., Garner, O. B., Bishop, J. R., Zhang, D. Y., Bush, K. T., Nigam, S. K., and Esko, J. D. (2010) PLoS One 5, e10691; Garner, O .B., Bush, K. T., Nigam, S .K., Yamaguchi, Y., Xu, D., Esko, J. D., and Nigam, S. K. (2011) Dev. Biol. 355, 394–403). Here, we analyzed the effect of deleting both Ndst2 and Ndst1. Whereas deletion of Ndst1 has no major effect on primary or secondary branching, deletion of Ndst2 appears to result in a mild increase in branching. When both genes were deleted, ductal growth was variably diminished (likely due to variable Cre-recombinase activity), but an overabundance of branched structures was evident irrespective of the extent of gland growth or postnatal age. “Hyperbranching” is an unusual phenotype. The effects on N-sulfation and growth factor binding were confirmed biochemically. The results indicate that N-sulfation or a factor requiring N-sulfation regulates primary and secondary branching events in the developing mammary gland. Together with previous work, the data indicate that different stages of ductal branching and lobuloalveolar formation are regulated by distinct sets of heparan sulfate biosynthetic enzymes in an appropriate growth factor context.     

Analysis of Drosophila Glucuronyl C5-Epimerase: IMPLICATIONS FOR DEVELOPMENTAL ROLES OF HEPARAN SULFATE SULFATION COMPENSATION AND 2-O-SULFATED GLUCURONIC ACID*

During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.     

Stage-dependent regulation of mammary ductal branching by heparan sulfate and HGF-cMet signaling

Specific interactions of growth factors with heparan sulfate may function as "switches" to regulate stages of branching morphogenesis in developing mammalian organs, such as breast, lung, salivary gland and kidney, but the evidence derives mostly from studies of explanted tissues or cell culture (Shah et al., 2004). We recently provided in vivo evidence that inactivation of Ndst1, the predominant N-deacetylase/N-sulfotransferase gene essential for the formation of mature heparan sulfate, results in a highly specific defect in murine lobuloalveolar development (Crawford et al., 2010). Here, we demonstrate a highly penetrant dramatic defect in primary branching by mammary epithelial-specific inactivation of Ext1, a subunit of the copolymerase complex that catalyzes the formation of the heparan sulfate chain. In contrast to Ext1 deletion, inactivation of Hs2st (which encodes an enzyme required for 2-O-sulfation of uronic acids in heparan sulfate) did not inhibit ductal formation but displayed markedly decreased secondary and ductal side-branches as well as fewer bifurcated terminal end buds. Targeted conditional deletion of c-Met, the receptor for HGF, in mammary epithelial cells showed similar defects in secondary and ductal side-branching, but did not result in any apparent defect in bifurcation of terminal end buds. Although there is published evidence indicating a role for 2-O sulfation in HGF binding, primary epithelial cells isolated from Hs2st conditional deletions were able to activate Erk in the presence of HGF and there appeared to be only a slight reduction in HGF-mediated c-Met phosphorylation in these cells compared to control. Thus, both c-Met and Hs2st play important, but partly independent, roles in secondary and ductal side-branching. When considered together with previous studies of Ndst1-deficient glands, the data presented here raise the possibility of partially-independent regulation by heparan sulfate-dependent pathways of primary ductal branching, terminal end bud bifurcation, secondary branching, ductal side-branching and lobuloalveolar formation.     

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.     

Heparan sulfate 3-O-sulfation: A rare modification in search of a function

Many protein ligands bind to heparan sulfate, which results in their presentation, protection, oligomerization or conformational activation. Binding depends on the pattern of sulfation and arrangement of uronic acid epimers along the chains. Sulfation at the C3 position of glucosamine is a relatively rare, yet biologically significant modification, initially described as a key determinant for binding and activation of antithrombin and later for infection by type I herpes simplex virus. In mammals, a family of seven heparan sulfate 3-O-sulfotransferases installs sulfate groups at this position and constitutes the largest group of sulfotransferases involved in heparan sulfate formation. However, to date very few proteins or biological systems have been described that are influenced by 3-O-sulfation. This review describes our current understanding of the prevalence and structure of 3-O-sulfation sites, expression and substrate specificity of the 3-O-sulfotransferase family and the emerging roles of 3-O-sulfation in biology.     

Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl^?) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.     

Heparin trisaccharides with nonreducing 2-amino-2-deoxy-α-d-glucopyranosyl end-groups suitable as substrates for catabolic enzymes

Heparin trisaccharides having the sequence O-(2-amino-2-deoxy-α-d-glucopyranosyl)-(1→4)-O-α-l-idopyranosyluronic acid-(1→4)-2,5-anhydro-d-[1-³H]mannitol have been prepared, as substrate models for studying sulfatases of heparan sulfate catabolism, by α-l-iduronidase cleavage of previously reported heparin tetrasaccharides, with additional chemical and enzymic modification as required. Three series are described, including isomeric sulfate esters of that trisaccharide with no N-substituent, with N-acetyl substitution, and with N-sulfate substitution. New features of the substrate specificity of the hydrolases used, including iduronate sulfatase, α-l-iduronidase, glucosamine 6-sulfate sulfatase, and heparin sulfamidase, were observed, and simple procedures for partial purification of these hydrolases are reported. The structures assigned to the trisaccharides are supported by the mode of preparation, reactions, regularities in electrophoretic behavior, and identities of the products of deamination.     

Vascular biomechanical properties in mice with smooth muscle specific deletion of Ndst1

Heparan sulfate proteoglycans act as co-receptors for many chemokines and growth factors. The sulfation pattern of the heparan sulfate chains is a critical regulatory step affecting the binding of chemokines and growth factors. N-deacetylase-N-sulfotransferase1 (Ndst1) is one of the first enzymes to catalyze sulfation. Previously published work has shown that HSPGs alter tangent moduli and stiffness of tissues and cells. We hypothesized that loss of Ndst1 in smooth muscle would lead to significant changes in heparan sulfate modification and the elastic properties of arteries. In line with this hypothesis, the axial tangent modulus was significantly decreased in aorta from mice lacking Ndst1 in smooth muscle (SM22αcre⁺Ndst1^?/?, p < 0.05, n = 5). The decrease in axial tangent modulus was associated with a significant switch in myosin and actin types and isoforms expressed in aorta and isolated aortic vascular smooth muscle cells. In contrast, no changes were found in the compliance of smaller thoracodorsal arteries of SM22αcre⁺Ndst1^?/? mice. In summary, the major findings of this study were that targeted ablation of Ndst1 in smooth muscle cells results in altered biomechanical properties of aorta and differential expression of myosin and actin types and isoforms.     

The Role of Drosophila Heparan Sulfate 6-O-Endosulfatase in Sulfation Compensation

The biosynthesis of heparan sulfate proteoglycans is tightly regulated by multiple feedback mechanisms, which support robust developmental systems. One of the regulatory network systems controlling heparan sulfate (HS) biosynthesis is sulfation compensation. A previous study using Drosophila HS 2-O- and 6-O-sulfotransferase (Hs2st and Hs6st) mutants showed that loss of sulfation at one position is compensated by increased sulfation at other positions, supporting normal FGF signaling. Here, we show that HS sulfation compensation rescues both Decapentaplegic and Wingless signaling, suggesting a universal role of this regulatory system in multiple pathways in Drosophila. Furthermore, we identified Sulf1, extracellular HS 6-O-endosulfatase, as a novel component of HS sulfation compensation. Simultaneous loss of Hs2st and Sulf1 led to 6-O-oversulfation, leading to patterning defects, overgrowth, and lethality. These phenotypes are caused at least partly by abnormal up-regulation of Hedgehog signaling. Thus, sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1.     

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.     

Basement membrane glycosaminoglycans: Examination of several membranes and evaluation of the effect of sonic treatment

The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment.     

Genetic epistasis between heparan sulfate and FGF-Ras signaling controls lens development

Vertebrate lens development depends on a complex network of signaling molecules to coordinate cell proliferation, migration and differentiation. In this study, we have investigated the role of heparan sulfate in lens specific signaling by generating a conditional ablation of heparan sulfate modification genes, Ndst1 and Ndst2. In this mutant, N-sulfation of heparan sulfate was disrupted after the lens induction stage, resulting in reduced lens cell proliferation, increased cell death and defective lens fiber differentiation in later lens development. The loss of Ndst function also prevented the assembly of Fgf/Fgfr complexes on the lens cell surface and disrupted ERK signaling within the lens. We further demonstrated that Ndst mutation completely inhibited the FGF1 and Fgf3 overexpression phenotypes, but Kras reactivation was sufficient to reverse the Ndst deficient lens differentiation defect. The epistatic relationship between Ndst and FGF–Ras signaling demonstrates that FGF signaling is the predominant signaling pathway controlled by Ndst in lens development.     

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.     

Inhibitory Peptides of the Sulfotransferase Domain of the Heparan Sulfate Enzyme,N-Deacetylase-N-sulfotransferase-1

N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.     

Heparan Sulfate Containing Unsubstituted Glucosamine Residues: BIOSYNTHESIS AND HEPARANASE-INHIBITORY ACTIVITY*

Degradation of heparan sulfate (HS) in the extracellular matrix by heparanase is linked to the processes of tumor invasion and metastasis. Thus, a heparanase inhibitor can be a potential anticancer drug. Because HS with unsubstituted glucosamine residues accumulates in heparanase-expressing breast cancer cells, we assumed that these HS structures are resistant to heparanase and can therefore be utilized as a heparanase inhibitor. As expected, chemically synthetic HS-tetrasaccharides containing unsubstituted glucosamine residues, GlcAβ1–4GlcNH₃⁺(6-O-sulfate)α1–4GlcAβ1–4GlcNH₃⁺(6-O-sulfate), inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro. Bifunctional NDST-1 (N-deacetylase/N-sulfotransferase-1) catalyzes the modification of N-acetylglucosamine residues within HS chains, and the balance of N-deacetylase and N-sulfotransferase activities of NDST-1 is thought to be a determinant of the generation of unsubstituted glucosamine. We also report here that EXTL3 (exostosin-like 3) controls N-sulfotransferase activity of NDST-1 by forming a complex with NDST-1 and contributes to generation of unsubstituted glucosamine residues.     

Heparan sulfate biosynthesis by embryonic tissues and primary fibroblast populations.

Fibroblasts from cornea, heart, and skin of day 14 embryonic chicks demonstrate the ability to make heparan sulfate-like polysaccharide when examined during the 10 hr period immediately following their removal from the embryo. Both the whole tissues from which these fibroblasts are isolated and the fibroblasts grown for 2–5 weeks in vitro also synthesize heparan sulfate. During their first few days in vitro, the three fibroblast populations display increasing rates of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporation into glycosaminoglycans and sharp fluctuations of those rates, yet the percentage of total [³⁵S]-sulfate incorporated into heparan sulfate-like polysaccharide and the distribution of this polysaccharide between cells and nutrient medium do not change significantly. During their first 48 hr in vitro, skin fibroblasts, but not those from cornea or heart, show steadily decreasing discrepancies between the proportions of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporated into heparan sulfate, suggesting a sharp decline in the synthesis of nonsulfated glycosaminoglycans. These data support the hypothesis of Kraemer than many cell-types in vivo may normally make heparan sulfate. The data largely eliminate the hypothesis that the biosynthesis of this polysaccharide is selectively stimulated as embryonic cells adapt to growth in vitro.     

Glycosaminoglycan synthesis by glomeruli in vivo and in vitro

The distribution of glycosaminoglycans in disrupted glomerular fractions was studied using ³⁵SO₄-labeling in vivo and in vitro. The majority of ³⁵S of isolated glomerular basement membrane was found in heparan sulfate after in vivo and in vitro pulses, although the absolute proportion and the degrees of N-sulfation and N-acetylation varied with the conditions of exposure. Varying amounts of chondroitin sulfate and dermatan sulfate were found in the glomerular basement membrane fraction and larger proportions of both of these glycosaminoglycans as well as of heparan sulfate were found in various glomerular fractions. Glomerular glycosaminoglycans distribution studies must take into account the experimental conditions. Basement membrane-like components of the glomerulus such as the mesangial matrix may have varying glycosaminoglycan composition which may be found in association with glomerular basement membrane fractions.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum: STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF CATALYTIC NITROGEN-SULFUR BOND CLEAVAGE*

Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides.