Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.     

Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol splitting

Heparanase is an endo-beta-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O- and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified D-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones.     

Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.     

Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Characterizing the non-reducing end structure of heparan sulfate

The reducing end of heparan sulfate has been known for a long time, but information on the non-reducing end has been lacking. Recent studies indicate that the non-reducing end of heparan sulfate might be the place where fibroblast growth factor signaling complex forms. The non-reducing end also changes with heparanase digestion and, thus, might serve as a marker for tumor pathology. Using high performance liquid chromatography-coupled mass spectrometry, we have identified and characterized the non-reducing end of bovine kidney heparan sulfate. We find that the non-reducing end region is highly sulfated and starts with a glucuronic acid (GlcA) residue. The likely sequence of the non-reducing end hexasaccharides is GlcA-GlcNS6S-UA+/-2S-GlcNS+/-6S-Ido2S-GlcNS+/-6S (where GlcNS is N-sulfate-D-glucosamine, S is sulfate, UA is uronic acid, and Ido is iduronic acid). Our data suggests that the non-reducing end of bovine kidney heparan sulfate is not trimmed by heparanase and is capable of supporting fibroblast growth factor signaling complex formation.     

Antithrombin-binding octasaccharides and role of extensions of the active pentasaccharide sequence in the specificity and strength of interaction. Evidence for very high affinity induced by an unusual glucuronic acid residue

The antithrombotic activity of low molecular weight heparins (LMWHs) is largely associated with the antithrombin (AT)-binding pentasaccharide sequence AGA(*)IA (GlcN(NAc/NS,6S)-GlcA-GlcN(NS,3,6S)-IdoUA(2S)-GlcN(NS,6S)). The location of the AGA(*)IA sequences along the LMWH chains is also expected to influence binding to AT. This study was aimed at investigating the role of the structure and molecular conformation of different disaccharide extensions on both sides of the AGA(*)IA sequence in modulating the affinity for AT. Four high purity octasaccharides isolated by size exclusion chromatography, high pressure liquid chromatography, and AT-affinity chromatography from the LMWH enoxaparin were selected for the study. All the four octasaccharides terminate at their nonreducing end with 4,5-unsaturated uronic acid residues (DeltaU). In two octasaccharides, AGA(*)IA was elongated at the reducing end by units IdoUA(2S)-GlcN(NS,6S) (OCTA-1) or IdoUA-GlcN(NAc,6S) (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA(*)IA was elongated at the nonreducing side by units GlcN(NS,6S)-IdoUA and GlcN(NS,6S)-GlcA, respectively. Extensions increased the affinity for AT of octasaccharides with respect to pentasaccharide AGA(*)IA, as also confirmed by fluorescence titration. Two-dimensional NMR and docking studies clearly indicated that, although elongation of the AGA(*)IA sequence does not substantially modify the bound conformation of the AGA(*)IA segment, extensions promote additional contacts with the protein. It should be noted that, as not previously reported, the unusual GlcA residue that precedes the AGA(*)IA sequence in OCTA-4 induced an unexpected 1 order of magnitude increase in the affinity to AT with respect to its IdoUA-containing homolog OCTA-3. Such a residue was found to orientate its two hydroxyl groups at close distance to residues of the protein. Besides the well established ionic interactions, nonionic interactions may thus contribute to strengthen oligosaccharide-AT complexes.     

Preparation of completely 6-O-desulfated heparin and its ability to enhance activity of basic fibroblast growth factor

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or (13)C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay.     

Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.     

Conformational transitions induced in heparin octasaccharides by binding with antithrombin III

The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Testosterone-induced growth of S115 mouse mammary tumor cells is dependent on heparan sulfate

The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

Demonstration of the pleiotrophin-binding oligosaccharide sequences isolated from chondroitin sulfate/dermatan sulfate hybrid chains of embryonic pig brains

Mammalian brains contain significant amounts of chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid chains. CS/DS chains isolated from embryonic pig brains (E-CS/DS) promote the outgrowth of neurites in embryonic mouse hippocampal neurons in culture by interacting with pleiotrophin (PTN), a heparin-binding growth factor. Here, we analyzed oligosaccharides isolated from E-CS/DS, which showed that octasaccharides were the minimal size capable of interacting with PTN at a physiological salt concentration. Five and eight sequences were purified from fluorescently labeled PTN-bound and -unbound octasaccharide fractions, respectively, by enzymatic digestion followed by PTN-affinity chromatography. Their sequences were determined by enzymatic digestion in conjunction with high performance liquid chromatography, revealing a critical role for oversulfated D and/or iD disaccharides in the low yet significant affinity for PTN, which is required for neuritogenesis. The critical D and iD units are GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate) and IdoUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate), respectively, where IdoUA represents L-iduronic acid. In contrast, high affinity interactions with PTN required decasaccharides with E units (GlcUAbeta1-3GalNAc(4, 6-O-disulfate)), B units (GlcUA(2-O-sulfate)beta1-3GalNAc(4-O-sulfate)), and/or their IdoUA-containing counterparts (iE and iB) in addition to D/iD units, although the biological significance of such strong interactions remains to be investigated. Thus, chain size and composition are crucial to the interaction with PTN, and PTN binds to multiple sequences in E-CS/DS chains with distinct affinity. Notably, not only heparan sulfate but also CS/DS hybrid chain structures of mammalian brains contain a high degree of microheterogeneity with a cluster of oversulfated disaccharides and appear to play roles in regulating the functions of PTN.     

Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate

The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]₂-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and trisulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluoresence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.Abbreviations aFGF,bFGF acidic and basic fibroblast growth factor - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - U,2S-(14)-GlcNS,6S O--L-ido(ene-pyranosyluronic acid 2-O-sulfate)-(14)-2-sulfoamino-2-deoxy-D-glucose-6-O-sulfate - U-(14)-GlcNS,6S O-(ene-pyranosyluronic acid)-(14)-2-sulfoamino-2-deoxy-D-glucose-6-O-sulfate - IdoUA iduronic acid - GlcUA glucuronic acid - GlyUA uronic acid; GlcNAcN-acetylglycosamine - GlcNS N-sulfated glucosamine - GlcNS,6S N,6-disulfated glucosamine - Gal galactose - Xyl xylose - Ser serine - HS heparan Sulfate     

Chondroitinase-mediated degradation of rare 3-O-sulfated glucuronic acid in functional oversulfated chondroitin sulfate K and E

Chondroitin sulfate K (CS-K) from king crab cartilage rich in rare 3-O-sulfated glucuronic acid (GlcUA(3S)) displayed neuritogenic activity and affinity toward various growth factors like CS-E from squid cartilage. CS-K-mediated neuritogenesis of mouse hippocampal neurons in culture was abolished by digestion with chondroitinase (CSase) ABC, indicating the possible involvement of GlcUA(3S). However, identification of GlcUA(3S) in CS chains by conventional high performance liquid chromatography has been hampered by its CSase ABC-mediated degradation. To investigate the degradation process, an authentic CS-E tetrasaccharide, Delta4,5HexUA-GalNAc(4S)-GlcUA(3S)-GalNAc(4S), was digested with CSase ABC, and the end product was identified as GalNAc(4S) by electrospray ionization mass spectrometry (ESI-MS). Putative GalNAc(6S) and GalNAc(4S,6S), derived presumably from GlcUA(3S)-GalNAc(6S) and GlcUA(3S)-GalNAc(4S,6S), respectively, were also detected by ESI-MS in the CSase ABC digest of a CS-E oligosaccharide fraction resistant to CSases AC-I and AC-II. Intermediates during the CSase ABC-mediated degradation of Delta4,5HexUA(3S)-GalNAc(4S) to GalNAc(4S) were identified through ESI-MS of a partial CSase ABC digest of a CS-K tetrasaccharide, GlcUA(3S)-GalNAc(4S)-GlcUA(3S)-GalNAc(4S), and the conceivable mechanism behind the degradation of the GlcUA(3S) moiety was elucidated. Although a fucose branch was also identified in CS-K, defucosylated CS-K exhibited greater neuritogenic activity than the native CS-K, excluding the possibility of the involvement of fucose in the activity. Rather, (3S)-containing disaccharides are likely involved. These findings will enable us to detect GlcUA(3S)-containing disaccharides in CS chains to better understand CS-mediated biological processes.     

Heparan sulfate proteoglycans and heparanase--partners in osteolytic tumor growth and metastasis.

This review summarizes a series of studies demonstrating that heparan sulfate proteoglycans act to promote the growth and metastasis of myeloma and breast tumors, two tumors that home to, and grow within, bone. Much of the growth-promoting effect of proteoglycans in these tumors may reside in the shed form of syndecan-1 that acts to favorably condition the tumor microenvironment. Moreover, the interplay between heparan sulfate and the extracellular enzyme heparanase-1 also has important regulatory implications. Recent studies indicate that the activity of heparanase, which likely releases heparin sulfate-bound growth factors and generates highly active heparan sulfate fragments, also promotes growth and metastasis of myeloma and breast tumors. Understanding the role of heparan sulfate and heparanase in the regulation of tumor behavior may lead to new therapeutic approaches for treating cancer.     

Synthesis of a tetrasaccharide substrate of heparanase

A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely β-d-GlcA(2S)-(1→4)-α-d-GlcN(NS,6S)-(1→4)-β-d-GlcA-(1→4)-α-d-GlcN(NS,6S)-OMe, was synthesized in a convergent manner via coupling of a pair of the disaccharide building blocks as a key step.     

The disaccharide repeating-units of heparan sulfate

Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.     

Downregulating the expression of heparanase inhibits the invasion, angiogenesis and metastasis of human hepatocellular carcinoma

Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC.     

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-β-D-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.